Compounds and methods for modulating expression of gccr

ABSTRACT

The present disclosure describes short antisense compounds, including such compounds comprising chemically-modified high-affinity monomers 8-16 monomers in length. Certain such short antisense compound are useful for the reduction of target nucleic acids and/or proteins in cells, tissues, and animals with increased potency and improved therapeutic index. Thus, provided herein are short antisense compounds comprising high-affinity nucleotide modifications useful for reducing a target RNA in vivo. Such short antisense compounds are effective at lower doses than previously described antisense compounds, allowing for a reduction in toxicity and cost of treatment. In addition, the described short antisense compounds have greater potential for oral dosing.

This application is a continuation-in-part of PCT ApplicationPCT/US2007/061183, filed Jan. 27, 2007, and hereby claims benefit ofpriority to that PCT Application under 35 U.S.C. § 120. This applicationalso claims the benefit of priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Serial Nos 60/746,631, filed May 5, 2006,60/747,059, filed May 11, 2006; 60/805,660, filed Jun. 23, 2006, and60/864,554, filed Nov. 6, 2006. Each of those applications, their entirecontents, and that of all references cited therein are herebyincorporated by reference in their entirety for any and all purposes.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCORE0061WO12SEQ.TXT, created on May 7, 2007 which is 700 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND

Targeting disease-causing gene sequences was first suggested nearly 40years ago (Belikova et al., Tet. Lett., 1967, 37, 3557-3562), andantisense activity was demonstrated in cell culture a decade later(Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A., 1978, 75, 280-284). Oneadvantage of antisense technology in the treatment of a disease orcondition that stems from a disease-causing gene is that it is a directgenetic approach that has the ability to modulate expression of specificdisease-causing genes.

Generally, the principle behind antisense technology is that anantisense compound hybridizes to a target nucleic acid and effectsmodulation of gene expression activity or function, such astranscription, translation or splicing. The modulation of geneexpression can be achieved by, for example, target degradation oroccupancy-based inhibition. An example of modulation of RNA targetfunction by degradation is RNase H-based degradation of the target RNAupon hybridization with a DNA-like antisense compound. Another exampleof modulation of gene expression by target degradation is RNAinterference (RNAi). RNAi is a form of antisense-mediated gene silencinginvolving the introduction of dsRNA leading to the sequence-specificreduction of targeted endogenous mRNA levels. Sequence-specificity makesantisense compounds extremely attractive as tools for target validationand gene functionalization, as well as research tools for identifyingand characterizing nucleases and as therapeutics to selectively modulatethe expression of genes involved in the pathogenesis of any one of avariety of diseases.

Antisense technology is an effective means for reducing the expressionof one or more specific gene products and can therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications. Chemically modified nucleosides are routinely used forincorporation into antisense compounds to enhance one or moreproperties, such as nuclease resistance, pharmacokinetics or affinityfor a target RNA.

Despite the expansion of knowledge since the discovery of antisensetechnology, there remains an unmet need for antisense compounds withgreater efficacy, reduced toxicity and lower cost. Until the presentdisclosure, high-affinity modifications have not been employed in thedesign of short antisense compounds for reducing target RNA in vivo.This is because of concerns regarding the degree of target specificitythat a sequence 15 nucleotides or shorter would have when employed toreduce target in a living system. Previous studies have described thatgreater specificity, and therefore greater potential for potency, isachieved by antisense compounds between 16 and 20 nucleobases in length.

The present disclosure describes incorporation of chemically-modifiedhigh-affinity nucleotides into antisense compounds allows for shortantisense compounds about 8-16 nucleobases in length useful in thereduction of target RNAs in animals with increased potency and improvedtherapeutic index. Thus, provided herein are short antisense compoundscomprising high-affinity nucleotide modifications useful for reducing atarget RNA in vivo. Such short antisense compounds are effective atlower doses than previously described antisense compounds, allowing fora reduction in toxicity and cost of treatment.

SUMMARY

Disclosed herein are short antisense compounds and methods of using saidcompounds to reduce target RNA expression in cells or tissues. Incertain embodiments, provided herein is a method of reducing expressionof a target in an animal, comprising administering to the animal a shortantisense compound targeted to a nucleic acid of such target. In certainembodiments, shorts antisense compounds are oligonucleotide compounds.In certain embodiments short antisense oligonucleotides are about 8 to16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to14 nucleotides in length and comprises a gap region flanked on each sideby a wing, wherein each wing independently consists of 1 to 3nucleotides. Preferred motifs include but are not limited to wing-deoxygap-wing motifs selected from 3-10-3, 2-10-3, 2-10-2, 1-10-1, 2-8-2,1-8-1, 3-6-3 or 1-6-1. In a preferred embodiment, the short antisenseoligonucleotide comprise at least one high-affinity modification. In afurther embodiment, the high-affinity modification includeschemically-modified high-affinity nucleotides. In a preferredembodiment, each wing independently consists of 1 to 3 high-affinitymodified nucleotides. In one embodiment the high affinity modifiednucleotides are sugar-modified nucleotides.

In certain embodiments short antisense compounds exhibit greater uptakein the gut as compared to antisense compounds of greater length. Thus,also provided herein are methods of reducing a target in an animal,comprising orally administering the short antisense compounds of thepresent invention.

In certain embodiments, short antisense compounds are targeted to anucleic acid encoding a protein selected from ApoB, SGLT2, PCSK9, SOD1,CRP, GCCR, GCGR, DGAT2, PTP1B and PTEN.

Further provided are methods of treating a metabolic disorder in ananimal, comprising administering to an animal in need of such therapy ashort antisense compound targeted to a nucleic acid involved inregulating glucose metabolism or clearance, lipid metabolism,cholesterol metabolism, or insulin signaling.

Also provided are methods of increasing insulin sensitivity, decreasingblood glucose or decreasing HbA_(1c) in an animal, comprisingadministering to said animal a short antisense compound targeted to anucleic acid encoding a target involved in regulating glucose metabolismor clearance, lipid metabolism, cholesterol metabolism, or insulinsignaling.

Further provided are methods of decreasing total serum cholesterol,serum LDL, serum VLDL, serum HDL, serum triglycerides, serumapolipoprotein(a) or free fatty acids in an animal, comprisingadministering to said animal a short antisense compound targeted to anucleic acid encoding a target that is involved in regulating glucosemetabolism or clearance, lipid metabolism, cholesterol metabolism, orinsulin signaling, wherein said short antisense compound is 8 to 16nucleotides in length and comprises a gap region flanked on each side bya wing, wherein each wing independently consists of 1 to 3 high-affinitymodified nucleotides.

Certain targets involved in regulating glucose metabolism or clearance,lipid metabolism, cholesterol metabolism, or insulin signaling include,but are not limited to, GCGR and ApoB-100. Thus, provided are shortantisense compounds targeting nucleic acids encoding GCGR and ApoB-100and methods of reducing expression of said targets and/or target nucleicacids in animal. In addition, provided is the use of short antisensecompounds targeting nucleic acids encoding GCGR, and ApoB-100 for thetreatment of a metabolic or cardiovascular disease or condition.

In certain embodiments, short antisense compounds further comprise aconjugate group. Conjugate groups include, but are not limited to, C₁₆and cholesterol.

In certain embodiments short antisense compounds comprise at least onemodified nucleobase, internucleoside linkage or sugar moiety. In certainembodiments, such modified internucleoside linkage is a phosphorothioateinternucleoside linkage. In certain embodiments, each internucleosidelinkage is a phosphorothioate internucleoside linkage.

In certain embodiments, short antisense compounds comprise at least onehigh affinity modification. In certain such embodiments, thehigh-affinity modification is a chemically-modified high-affinitynucleotide. In certain embodiments, chemically-modified high affinitynucleotides are sugar-modified nucleotides. In certain embodiments, atleast one of the sugar-modified nucleotides comprises a bridge betweenthe 4′ and the 2′ position of the sugar. Each of the sugar-modifiednucleotides is, independently, in the β-D or α-L sugar conformation. Incertain embodiments, each of said high-affinity modified nucleotidesconfers a T_(m) of at least 1 to 4 degrees per nucleotide. In certainembodiments, each of said sugar-modified nucleotides comprises a2′-substituent group that is other than H or OH. Such sugar-modifiednucleotides include those having a 4′ to 2′ bridged bicyclic sugarmoiety. In certain embodiments, each of the 2′-substituent groups is,independently, alkoxy, substituted alkoxy, or halogen. In certainembodiments, each of the 2′-substituent groups is OCH₂CH₂OCH₃ (2′-MOE).

In certain embodiments, short antisense compounds have one or moresugar-modified nucleotides comprising a bridge between the 4′ and 2′position of the sugar, wherein each of said bridges independentlycomprises from 2 to 4 linked groups independently selected from—[C(R₁)(R₂)]_(n)—, —C(R₁)═C(R₂)—, —C(R₁)═N—, —C(═NR₁)—, —C(═O)—,—C(═S)—, —O—, —Si(R₁)₂—, —S(═O)_(n)— and —N(R₁)—;

wherein

-   -   x is 0, 1, or 2;    -   n is 1, 2, 3, or 4;    -   each R₁ and R₂ is, independently, H, a protecting group,        hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂        alkenyl, substituted C₂-C₁₂ alkenyl, C_(2 The)-C₁₂ alkynyl,        substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀        aryl, heterocycle radical, substituted heterocycle radical,        heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,        substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁,        N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl        (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and        -   each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl,            substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂            alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀            aryl, substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted            acyl, a heterocycle radical, a substituted heterocycle            radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl or            a protecting group.

In one aspect, each of said bridges is, independently, —[C(R₁)(R₂)]_(n),—[C(R₁)(R₂)]_(n)—O—, —C(R₁R₂)—N(R₁)—O— or C(R₁R₂)—O—N(R₁)—. In anotheraspect, each of said bridges is, independently, 4′-(CH₂)₃-2′,4′-(CH₂)₂-2′, 4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R₁)-2′ and4′-CH₂—N(R₁)—O-2′- wherein each R₁ is, independently, H, a protectinggroup or C₁-C₁₂ alkyl.

In certain embodiments, provided herein are short antisense compoundsuseful in the reduction of targets and/or target RNAs associated withdisease states in animals. In certain embodiments, provided are methodsof using the short antisense compounds for reducing expression of atarget RNA in an animal. In certain embodiments, provided herein is theuse of a short antisense compound in the preparation of a medicament forthe treatment of a metabolic disorder in an animal. In certainembodiments, provided herein is the use of a short antisense compound inthe preparation of a medicament for increasing insulin sensitivity,decreasing blood glucose or decreasing HbA_(1c) in an animal. Alsoprovided is the use of a short antisense compound in the preparation ofa medicament for decreasing total serum cholesterol, serum LDL, serumVLDL, serum HDL, serum triglycerides, serum apolipoprotein(a) or freefatty acids in an animal.

In certain embodiments, short antisense compounds provided hereinexhibit equal or increased potency with regard to target RNA knockdownas compared to longer parent antisense oligonucleotide at least 20nucleotides in length. In certain embodiments, short antisense compoundsexhibit a faster onset of action (target RNA reduction) as compared tothe parent antisense oligonucleotide. In certain embodiments, increasedpotency is in the kidney. In certain embodiments, target RNA ispredominately expressed in the kidney. In certain embodiments, increasedpotency is in the liver. In certain embodiments, target RNA ispredominately expressed in the liver.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose. U.S. patent application Ser. Nos.10/712,795 and 10/200,710 are hereby expressly incorporated by referencein their entirety for any purpose.

A. DEFINITIONS

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, chemicalanalysis, pharmaceutical preparation, formulation and delivery, andtreatment of subjects. Certain such techniques and procedures may befound for example in “Carbohydrate Modifications in Antisense Research”Edited by Sangvi and Cook, American Chemical Society, Washington D.C.,1994; and “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., 18th edition, 1990; and which is hereby incorporated byreference for any purpose. Where permitted, all patents, applications,published applications and other publications and sequences from GenBankand other data bases referred to throughout in the disclosure herein areincorporated by reference in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, the term “nucleoside” means a glycosylamine comprising anucleobase and a sugar. Nucleosides includes, but are not limited to,naturally occurring nucleosides, abasic nucleosides, modifiednucleosides, and nucleosides having mimetic bases and/or sugar groups.

As used herein, the term “nucleotide” refers to a glycosamine comprisinga nucleobase and a sugar having a phosphate group covalently linked tothe sugar. Nucleotides may be modified with any of a variety ofsubstituents.

As used herein, the term “nucleobase” refers to the base portion of anucleoside or nucleotide. A nucleobase may comprise any atom or group ofatoms capable of hydrogen bonding to a base of another nucleic acid.

As used herein, the term “heterocyclic base moiety” refers to anucleobase comprising a heterocycle.

As used herein, the term “deoxyribonucleotide” means a nucleotide havinga hydrogen at the 2′ position of the sugar portion of the nucleotide.Deoxyribonucleotides may be modified with any of a variety ofsubstituents.

As used herein, the term “ribonucleotide” means a nucleotide having ahydroxy at the 2′ position of the sugar portion of the nucleotide.Ribonucleotides may be modified with any of a variety of substituents.

As used herein, the term “oligomeric compound” refers to a polymericstructure comprising two or more sub-structures and capable ofhybridizing to a region of a nucleic acid molecule. In certainembodiments, oligomeric compounds are oligonucleosides. In certainembodiments, oligomeric compounds are oligonucleotides. In certainembodiments, oligomeric compounds are antisense compounds. In certainembodiments, oligomeric compounds are antisense oligonucleotides. Incertain embodiments, oligomeric compounds are short antisense compounds.In certain embodiments, oligomeric compounds are short antisenseoligonucleotides. In certain embodiments, oligomeric compounds arechimeric oligonucleotides.

As used herein, the term “monomer” refers to a single unit of anoligomer. Monomers include, but are not limited to, nucleosides andnucleotides, whether naturally occurring or modified.

As used herein “oligonucleoside” refers to an oligonucleotide in whichthe internucleoside linkages do not contain a phosphorus atom.

As used herein, the term “oligonucleotide” refers to an oligomericcompound comprising a plurality of linked nucleotides. In certainembodiment, one or more nucleotides of an oligonucleotide is modified.In certain embodiments, an oligonucleotide comprises ribonucleic acid(RNA) or deoxyribonucleic acid (DNA). In certain embodiments,oligonucleotides are composed of naturally- and/ornon-naturally-occurring nucleobases, sugars and covalent internucleotidelinkages, and may further include non-nucleic acid conjugates.

As used herein “internucleotide linkage” refers to a covalent linkagebetween adjacent nucleotides.

As used herein, the term “monomeric linkage” refers to a covalentlinkage between two monmers. Monomeric linkages include, but are notlimited to internucleotide linkages and internucleoside linkages.

As used herein “naturally occurring internucleotide linkage” refers to a3′ to 5′ phosphodiester linkage.

As used herein, the term “antisense compound” refers to an oligomericcompound that is at least partially complementary to a target nucleicacid molecule to which it hybridizes. In certain embodiments, anantisense compound modulates (increases or decreases) expression of atarget nucleic acid. Antisense compounds include, but are not limitedto, compounds that are oligonucleotides, oligonucleosides,oligonucleotide analogs, oligonucleotide mimetics, and chimericcombinations of these. Consequently, while all antisense compounds areoligomeric compounds, not all oligomeric compounds are antisensecompounds.

As used herein, the term “antisense oligonucleotide” refers to anantisense compound that is an oligonucleotide.

As used herein, the term “parent antisense oligonucleotide” refers to anoligonucleotide 20 nucleotides in length having a deoxy gap regionhaving ten 2′-deoxyribonucleotides, flanked by a first and a second wingregion each having five 2′-O-(2-methoxyethyl) ribonucleotides (a 5-10-5MOE gapmer) and comprising the sequence of the corresponding shortantisense compound to which it is a parent.

As used herein, the term “short antisense compound” refers to anantisense compound about 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomers inlength. In certain embodiments, a short antisense compound has at leastone high-affinity modification.

As used herein, the term “short antisense oligonucleotide” or refers toan antisense oligonucleotide about 8, 9, 10, 11, 12, 13, 14, 15 or 16nucleotides in length. In certain embodiments, a short antisenseoligonucleotide has at least one high-affinity modification.

As used herein, the term “short gapmer” refers to a short antisenseoligonucleotide having a first and a second wing region eachindependently 1 to 3 nucleotides in length and a gap region 2 to 14nucleobase in length.

As used herein, the term “motif” refers to the pattern of unmodified andmodified nucleotides in a short antisense compound

As used herein, the term “chimeric antisense oligomer” refers to anantisense oligomeric compound, having at least one sugar, nucleobase orinternucleoside linkage that is differentially modified as compared toat least on other sugar, nucleobase or internucleoside linkage withinthe same antisense oligomeric compound. The remainder of the sugars,nucleobases and internucleoside linkages can be independently modifiedor unmodified, the same or different.

As used herein, the term “chimeric antisense oligonucleotide” refers toan antisense oligonucleotide, having at least one sugar, nucleobase orinternucleoside linkage that is differentially modified as compared toat least on other sugar, nucleobase or internucleoside linkage withinthe same antisense oligonucleotide. The remainder of the sugars,nucleobases and internucleoside linkages can be independently modifiedor unmodified, the same or different.

As used herein, the term “mixed-backbone antisense oligonucleotide”refers to an antisense oligonucleotide wherein at least oneinternucleoside linkage of the antisense oligonucleotide is differentfrom at least one other internucleotide linkage of the antisenseoligonucleotide.

As used herein, the term “target” refers to a protein, the modulation ofwhich is desired.

As used herein, the term “target gene” refers to a gene encoding atarget.

As used herein, the terms “target nucleic acid” and “nucleic acidmolecule encoding a target” refer to any nucleic acid molecule theexpression or activity of which is capable of being modulated by anantisense compound. Target nucleic acids include, but are not limitedto, RNA (including, but not limited to pre-mRNA and mRNA or portionsthereof) transcribed from DNA encoding a target, and also cDNA derivedfrom such RNA, and miRNA. For example, the target nucleic acid can be acellular gene (or mRNA transcribed from the gene) whose expression isassociated with a particular disorder or disease state, or a nucleicacid molecule from an infectious agent.

As used herein, the term “targeting” or “targeted to” refers to theassociation of an antisense compound to a particular target nucleic acidmolecule or a particular region of nucleotides within a target nucleicacid molecule.

As used herein, the term “5′ target site” refers to the nucleotide of atarget nucleic acid which is complementary to the 5′-most nucleotide ofa particular antisense compound.

As used herein, the term “3′ target site” refers to the nucleotide of atarget nucleic acid which is complementary to the 3′-most nucleotide ofa particular antisense compound.

As used herein, the term “target region,” refers to a portion of atarget nucleic acid to which one or more antisense compounds iscomplementary.

As used herein, the term “target segment” refers to a smaller orsub-portions of a region within a target nucleic acid.

As used herein, the term “nucleobase complementarity” refers to anucleobase that is capable of base pairing with another nucleobase. Forexample, in DNA, adenine (A) is complementary to thymine (T). Forexample, in RNA, adenine (A) is complementary to uracil (U). In certainembodiments, complementary nucleobase refers to a nucleobase of anantisense compound that is capable of base pairing with a nucleobase ofits target nucleic acid. For example, if a nucleobase at a certainposition of an antisense compound is capable of hydrogen bonding with anucleobase at a certain position of a target nucleic acid, then theposition of hydrogen bonding between the oligonucleotide and the targetnucleic acid is considered to be complementary at that nucleobase pair.

As used herein, the term “non-complementary nucleobase” refers to a pairof nucleobases that do not form hydrogen bonds with one another orotherwise support hybridization.

As used herein, the term “complementary” refers to the capacity of anoligomeric compound to hybridize to another oligomeric compound ornucleic acid through nucleobase complementarity. In certain embodiments,an antisense compound and its target are complementary to each otherwhen a sufficient number of corresponding positions in each molecule areoccupied by nucleobases that can bond with each other to allow stableassociation between the antisense compound and the target. One skilledin the art recognizes that the inclusion of mismatches is possiblewithout eliminating the ability of the oligomeric compounds to remain inassociation. Therefore, described herein are antisense compounds thatmay comprise up to about 20% nucleotides that are mismatched (i.e., arenot nucleobase complementary to the corresponding nucleotides of thetarget). Preferably the antisense compounds contain no more than about15%, more preferably not more than about 10%, most preferably not morethan 5% or no mismatches. The remaining nucleotides are nucleobasecomplementary or otherwise do not disrupt hybridization (e.g., universalbases). One of ordinary skill in the art would recognize the compoundsprovided herein are at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%complementary to a target nucleic acid.

As used herein, the term “mismatch” refers to a non-complementarynucleobase within a complementary oligomeric compound.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases). For example,the natural base adenine is nucleobase complementary to the naturalnucleobases thymidine and uracil which pair through the formation ofhydrogen bonds. The natural base guanine is nucleobase complementary tothe natural bases cytosine and 5-methyl cytosine. Hybridization canoccur under varying circumstances.

As used herein, the term “specifically hybridizes” refers to the abilityof an oligomeric compound to hybridize to one nucleic acid site withgreater affinity than it hybridizes to another nucleic acid site. Incertain embodiments, an antisense oligonucleotide specificallyhybridizes to more than one target site.

As used herein, “designing” or “designed to” refer to the process ofdesigning an oligomeric compound that specifically hybridizes with aselected nucleic acid molecule.

As used herein, the term “modulation” refers to a perturbation offunction or activity when compared to the level of the function oractivity prior to modulation. For example, modulation includes thechange, either an increase (stimulation or induction) or a decrease(inhibition or reduction) in gene expression. As further example,modulation of expression can include perturbing splice site selection ofpre-mRNA processing.

As used herein, the term “expression” refers to all the functions andsteps by which a gene's coded information is converted into structurespresent and operating in a cell. Such structures include, but are notlimited to the products of transcription and translation.

As used herein, “variant” refers to an alternative RNA transcript thatcan be produced from the same genomic region of DNA. Variants include,but are not limited to “pre-mRNA variants” which are transcriptsproduced from the same genomic DNA that differ from other transcriptsproduced from the same genomic DNA in either their start or stopposition and contain both intronic and exonic sequence. Variants alsoinclude, but are not limited to, those with alternate splice junctions,or alternate initiation and termination codons.

As used herein, “high-affinity modified monomer” refers to a monomerhaving at least one modified nucleobase, internucleoside linkage orsugar moiety, when compared to naturally occurring monomers, such thatthe modification increases the affinity of an antisense compoundcomprising the high-affinity modified monomer to its target nucleicacid. High-affinity modifications include, but are not limited to,monomers (e.g., nucleosides and nucleotides) comprising 2′-modifiedsugars.

As used herein, the term “2′-modified” or “2′-substituted” means a sugarcomprising substituent at the 2′ position other than H or OH.2′-modified monomers, include, but are not limited to, BNA's andmonomers (e.g., nucleosides and nucleotides) with 2′-substituents, suchas allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃,O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. Incertain embodiments, short antisense compounds comprise a 2′modifiedmonomer that does not have the formula 2′-O(CH₂)_(n)H, wherein n is oneto six. In certain embodiments, short antisense compounds comprise a2′modified monomer that does not have the formula 2′-OCH₃ In certainembodiments, short antisense compounds comprise a 2′modified monomerthat does not have the formula or, in the alternative, 2′-O(CH₂)₂OCH₃.

As used herein, the term “bicyclic nucleic acid” or “BNA” or “bicyclicnucleoside” or “bicyclic nucleotide” refers to a nucleoside ornucleotide wherein the furanose portion of the nucleoside includes abridge connecting two carbon atoms on the furanose ring, thereby forminga bicyclic ring system.

As used herein, unless otherwise indicated, the term “methyleneoxy BNA”alone refers to β-D-methyleneoxy BNA.

As used herein, the term “MOE” refers to a 2′-methoxyethyl substituent.

As used herein, the term “gapmer” refers to a chimeric oligomericcompound comprising a central region (a “gap”) and a region on eitherside of the central region (the “wings”), wherein the gap comprises atleast one modification that is different from that of each wing. Suchmodifications include nucleobase, monomeric linkage, and sugarmodifications as well as the absence of modification (unmodified). Thus,in certain embodiments, the nucleotide linkages in each of the wings aredifferent than the nucleotide linkages in the gap. In certainembodiments, each wing comprises nucleotides with high affinitymodifications and the gap comprises nucleotides that do not comprisethat modification. In certain embodiments the nucleotides in the gap andthe nucleotides in the wings all comprise high affinity modifications,but the high affinity modifications in the gap are different than thehigh affinity modifications in the wings. In certain embodiments, themodifications in the wings are the same as one another. In certainembodiments, the modifications in the wings are different from eachother. In certain embodiments, nucleotides in the gap are unmodified andnucleotides in the wings are modified. In certain embodiments, themodification(s) in each wing are the same. In certain embodiments, themodification(s) in one wing are different from the modification(s) inthe other wing. In certain embodiments, short antisense compounds aregapmers having 2′-deoxynucleotides in the gap and nucleotides withhigh-affinity modifications in the wing.

As used herein, the term “prodrug” refers to a therapeutic agent that isprepared in an inactive form that is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes or other chemicals and/or conditions.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts of active compounds that retain the desired biological activity ofthe active compound and do not impart undesired toxicological effectsthereto.

As used herein, the term “cap structure” or “terminal cap moiety” refersto chemical modifications, which have been incorporated at eitherterminus of an antisense compound.

As used herein, the term “prevention” refers to delaying or forestallingthe onset or development of a condition or disease for a period of timefrom hours to days, preferably weeks to months.

As used herein, the term “amelioration” refers to a lessening of atleast one indicator of the severity of a condition or disease. Theseverity of indicators may be determined by subjective or objectivemeasures which are known to those skilled in the art.

As used herein, the term “treatment” refers to administering acomposition of the invention to effect an alteration or improvement ofthe disease or condition. Prevention, amelioration, and/or treatment mayrequire administration of multiple doses at regular intervals, or priorto onset of the disease or condition to alter the course of the diseaseor condition. Moreover, a single agent may be used in a singleindividual for each prevention, amelioration, and treatment of acondition or disease sequentially, or concurrently.

As used herein, the term “pharmaceutical agent” refers to a substanceprovides a therapeutic benefit when administered to a subject.

As used herein, the term “therapeutically effective amount” refers to anamount of a pharmaceutical agent that provides a therapeutic benefit toan animal.

As used herein, “administering” means providing a pharmaceutical agentto an animal, and includes, but is not limited to administering by amedical professional and self-administering.

As used herein, the term “co-administration” refers to administration oftwo or more pharmaceutical agents to an animal. The two or morepharmaceutical agents may be in a single pharmaceutical composition, ormay be in separate pharmaceutical compositions. Each of the two or morepharmaceutical agents may be administered through the same or differentroutes of administration. Co-administration encompasses administrationin parallel or sequentially.

As used herein, the term “pharmaceutical composition” refers to amixture of substances suitable for administering to an individual. Forexample, a pharmaceutical composition may comprise an antisenseoligonucleotide and a sterile aqueous solution.

As used herein, the term “individual” refers to a human or non-humananimal selected for treatment or therapy.

As used herein, the term “animal” refers to a human or non-human animal,including, but not limited to, mice, rats, rabbits, dogs, cats, pigs,and non-human primates, including, but not limited to, monkeys andchimpanzees.

As used herein, the term “subject” refers to an animal, including, butnot limited to a human, to whom a pharmaceutical composition isadministered.

As used herein, the term “duration” refers to the period of time duringwhich an activity or event continues. In certain embodiments, theduration of treatment is the period of time during which doses of apharmaceutical agent are administered.

As used herein, the term “parenteral administration,” refers toadministration through injection or infusion. Parenteral administrationincludes, but is not limited to, subcutaneous administration,intravenous administration, or intramuscular administration.

As used herein, the term “subcutaneous administration” refers toadministration just below the skin. “Intravenous administration” meansadministration into a vein.

As used herein, the term “dose” refers to a specified quantity of apharmaceutical agent provided in a single administration. In certainembodiments, a dose may be administered in two or more boluses, tablets,or injections. For example, in certain embodiments, where subcutaneousadministration is desired, the desired dose requires a volume not easilyaccommodated by a single injection. In such embodiments, two or moreinjections may be used to achieve the desired dose. In certainembodiments, a dose may be administered in two or more injections tominimize injection site reaction in an individual.

As used herein, the term “dosage unit” refers to a form in which apharmaceutical agent is provided. In certain embodiments, a dosage unitis a vial comprising lyophilized antisense oligonucleotide. In certainembodiments, a dosage unit is a vial comprising reconstituted antisenseoligonucleotide.

As used herein, the term “pharmaceutical agent” refers to a substanceprovides a therapeutic benefit when administered to an individual. Forexample, in certain embodiments, an antisense oligonucleotide is apharmaceutical agent.

As used herein, the term “active pharmaceutical ingredient” refers tothe substance in a pharmaceutical composition that provides a desiredeffect.

As used herein, the term. “therapeutically effective amount” refers toan amount of a pharmaceutical agent that provides a therapeutic benefitto an individual. In certain embodiments, a therapeutically effectiveamount of an antisense compound is the amount that needs to beadministered to result in an observable benefit.

As used herein, the term “hypercholesterolemia” refers to a conditioncharacterized by elevated serum cholesterol.

As used herein, the term “hyperlipidemia” refers to a conditioncharacterized by elevated serum lipids.

As used herein, the term “hypertriglyceridemia” refers to a conditioncharacterized by elevated triglyceride levels.

As used herein, the term “non-familial hypercholesterolemia” refers to acondition characterized by elevated cholesterol that is not the resultof a single inherited gene mutation.

As used herein, the term “polygenic hypercholesterolemia” refers to acondition characterized by elevated cholesterol that results from theinfluence of a variety of genetic factors. In certain embodiments,polygenic hypercholesterolemia may be exacerbated by dietary intake oflipids.

As used herein, the term “familial hypercholesterolemia (FH)” refers toan autosomal dominant metabolic disorder characterized by a mutation inthe LDL-receptor (LDL-R) gene, markedly elevated LDL-C and prematureonset of atherosclerosis. A diagnosis of familial hypercholesterolemiais made when a individual meets one or more of the following criteria:genetic testing confirming 2 mutated LDL-receptor genes; genetic testingconfirming one mutated LDL-receptor gene; document history of untreatedserum LDL-cholesterol greater than 500 mg/dL; tendinous and/or cutaneousxanthoma prior to age 10 years; or, both parents have documentedelevated serum LDL-cholesterol prior to lipid-lowering therapyconsistent with heterozygous familial hypercholesterolemia.

As used herein, the term “homozygous familial hypercholesterolemia” or“HoFH” refers to a condition characterized by a mutation in bothmaternal and paternal LDL-R genes.

As used herein, the term “heterozygous familial hypercholesterolemia” or“HeFH” refers to a condition characterized by a mutation in either thematernal or paternal LDL-R gene.

As used herein, the term “mixed dyslipidemia” refers to a conditioncharacterized by elevated serum cholesterol and elevated serumtriglycerides.

As used herein, the term “diabetic dyslipidemia” or “Type II diabeteswith dyslipidemia” refers to a condition characterized by Type IIdiabetes, reduced HDL-C, elevated serum triglycerides, and elevatedsmall, dense LDL particles.

As used herein, the term “CHD risk equivalents,” refers to indicators ofclinical atherosclerotic disease that confer a high risk for coronaryheart disease. For example, in certain embodiments, CHD risk equivalentsinclude, without limitation, clinical coronary heart disease,symptomatic carotid artery disease, peripheral arterial disease, and/orabdominal aortic aneurysm.

As used herein, the term “non-alcoholic fatty liver disease (NAFLD)”refers to a condition characterized by fatty inflammation of the liverthat is not due to excessive alcohol use (for example, alcoholconsumption of over 20 g/day). In certain embodiments, NAFLD is relatedto insulin resistance and the metabolic syndrome.

As used herein, the term “non-alcoholic steatohepatitis (NASH)” refersto a condition characterized by inflammation and the accumulation of fatand fibrous tissue in the liver, that is not due to excessive alcoholuse. NASH is an extreme form of NAFLD.

As used herein, the term “major risk factors” refers to factors thatcontribute to a high risk for a particular disease or condition. Incertain embodiments, major risk factors for coronary heart diseaseinclude, without limitation, cigarette smoking, hypertension, low HDL-C,family history of coronary heart disease, and age.

As used herein, the term “CHD risk factors” refers to CHD riskequivalents and major risk factors.

As used herein, the term “coronary heart disease (CHD)” refers to anarrowing of the small blood vessels that supply blood and oxygen to theheart, which is often a result of atherosclerosis.

As used herein, the term “reduced coronary heart disease risk” refers toa reduction in the likelihood that a individual will develop coronaryheart disease. In certain embodiments, a reduction in coronary heartdisease risk is measured by an improvement in one or more CHD riskfactors, for example, a decrease in LDL-C levels.

As used herein, the term “atherosclerosis” refers to a hardening of thearteries affecting large and medium-sized arteries and is characterizedby the presence of fatty deposits. The fatty deposits are called“atheromas” or “plaques,” which consist mainly of cholesterol and otherfats, calcium and scar tissue, and damage the lining of arteries.

As used herein, the term “history of coronary heart disease” refers tothe occurrence of clinically evident coronary heart disease in themedical history of a individual or a individual's family member.

As used herein, the term “Early onset coronary heart disease” refers toa diagnosis of coronary heart disease prior to age 50.

As used herein, the term “statin intolerant individual” refers to aindividual who as a result of statin therapy experiences one or more ofcreatine kinase increases, liver function test abnormalities, muscleaches, or central nervous system side effects.

As used herein, the term “efficacy” refers to the ability to produce adesired effect. For example, efficacy of a lipid-lowering therapy may bereduction in the concentration of one or more of LDL-C, VLDL-C, IDL-C,non-HDL-C, ApoB, lipoprotein(a), or triglycerides.

As used herein, the term “acceptable safety profile” refers to a patternof side effects that is within clinically acceptable limits.

As used herein, the term “side effects” refers to physiologicalresponses attributable to a treatment other than desired effects. Incertain embodiments, side effects include, without limitation, injectionsite reactions, liver function test abnormalities, renal functionabnormalities, liver toxicity, renal toxicity, central nervous systemabnormalities, and myopathies. For example, increased aminotransferaselevels in serum may indicate liver toxicity or liver functionabnormality. For example, increased bilirubin may indicate livertoxicity or liver function abnormality.

As used herein, the term “injection site reaction” refers toinflammation or abnormal redness of skin at a site of injection in anindividual.

As used herein, the term “individual compliance” refers to adherence toa recommended or prescribed therapy by an individual.

As used herein, the term “lipid-lowering therapy” refers to atherapeutic regimen provided to a individual to reduce one or morelipids in a individual. In certain embodiments, a lipid-lowering therapyis provide to reduce one or more of ApoB, total cholesterol, LDL-C,VLDL-C, IDL-C, non-HDL-C, triglycerides, small dense LDL particles, andLp(a) in an individual.

As used herein, the term “lipid-lowering agent” refers to apharmaceutical agent provided to a individual to achieve a lowering oflipids in the individual. For example, in certain embodiments, alipid-lowering agent is provided to an individual to reduce one or moreof ApoB, LDL-C, total cholesterol, and triglycerides.

As used herein, the term “LDL-C target” refers to an LDL-C level that isdesired following lipid-lowering therapy.

As used herein, the term “comply” refers to the adherence with arecommended therapy by an individual.

As used herein, the term “recommended therapy” refers to a therapeuticregimen recommended by a medical professional for the treatment,amelioration, or prevention of a disease.

As used herein, the term “low LDL-receptor activity” refers toLDL-receptor activity that is not sufficiently high to maintainclinically acceptable levels of LDL-C in the bloodstream.

As used herein, the term “cardiovascular outcome” refers to theoccurrence of major adverse cardiovascular events.

As used herein, the term “improved cardiovascular outcome” refers to areduction in the occurrence of major adverse cardiovascular events, orthe risk thereof. Examples of major adverse cardiovascular eventsinclude, without limitation, death, reinfarction, stroke, cardiogenicshock, pulmonary edema, cardiac arrest, and atrial dysrhythmias.

As used herein, the term “surrogate markers of cardiovascular outcome”refers to indirect indicators of cardiovascular events, or the riskthereof. For example, surrogate markers of cardiovascular outcomeinclude carotid intimal media thickness (CIMT). Another example of asurrogate marker of cardiovascular outcome includes atheroma size.Atheroma size may be determined by intravascular ultrasound (IVUS).

As used herein, the term “increased HDL-C” refers to an increase inserum HDL-C in an individual over time.

As used herein, the term “lipid-lowering” refers to a reduction in oneor more serum lipids in an individual over time.

As used herein, the term “metabolic disorder” refers to a conditioncharacterized by an alteration or disturbance in metabolic function.“Metabolic” and “metabolism” are terms well know in the art andgenerally include the whole range of biochemical processes that occurwithin a living organism. Metabolic disorders include, but are notlimited to, hyperglycemia, prediabetes, diabetes (type I and type II),obesity, insulin resistance and metabolic syndrome.

As used herein, the term “metabolic syndrome” refers to a clustering oflipid and non-lipid cardiovascular risk factors of metabolic origin. Ithas been closely linked to the generalized metabolic disorder known asinsulin resistance. The National Cholesterol Education Program (NCEP)Adult Treatment Panel III (ATPIII) established criteria for diagnosis ofmetabolic syndrome when three or more of five risk determinants arepresent. The five risk determinants are abdominal obesity defined aswaist circumference of greater than 102 cm for men or greater than 88 cmfor women, triglyceride levels greater than or equal to 150 mg/dL, HDLcholesterol levels of less than 40 mg/dL for men and less than 50 mg/dLfor women, blood pressure greater than or equal to 130/85 mm Hg andfasting glucose levels greater than or equal to 110 mg/dL. Thesedeterminants can be readily measured in clinical practice (JAMA, 2001,285: 2486-2497).

The term “alkyl,” as used herein, refers to a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include, but are not limited to, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred. The term “lower alkyl” asused herein includes from 1 to about 6 carbon atoms. Alkyl groups asused herein may optionally include one or more further substituentgroups.

The term “alkenyl,” as used herein, refers to a straight or branchedhydrocarbon chain radical containing up to twenty four carbon atoms andhaving at least one carbon-carbon double bond. Examples of alkenylgroups include, but are not limited to, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like.Alkenyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkenyl groups as used herein may optionallyinclude one or more further substituent groups.

The term “alkynyl,” as used herein, refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms and havingat least one carbon-carbon triple bond. Examples of alkynyl groupsinclude, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and thelike. Alkynyl groups typically include from 2 to about 24 carbon atoms,more typically from 2 to about 12 carbon atoms with from 2 to about 6carbon atoms being more preferred. Alkynyl groups as used herein mayoptionally include one or more further substitutent groups.

The term “aminoalkyl” as used herein, refers to an amino substitutedalkyl radical. This term is meant to include C₁-C₁₂ alkyl groups havingan amino substituent at any position and wherein the alkyl groupattaches the aminoalkyl group to the parent molecule. The alkyl and/oramino portions of the aminoalkyl group can be further substituted withsubstituent groups.

The term “aliphatic,” as used herein, refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms whereinthe saturation between any two carbon atoms is a single, double ortriple bond. An aliphatic group preferably contains from 1 to about 24carbon atoms, more typically from 1 to about 12 carbon atoms with from 1to about 6 carbon atoms being more preferred. The straight or branchedchain of an aliphatic group may be interrupted with one or moreheteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Suchaliphatic groups interrupted by heteroatoms include without limitationpolyalkoxys, such as polyalkylene glycols, polyamines, and polyimines.Aliphatic groups as used herein may optionally include furthersubstitutent groups.

The term “alicyclic” or “alicyclyl” refers to a cyclic ring systemwherein the ring is aliphatic. The ring system can comprise one or morerings wherein at least one ring is aliphatic. Preferred alicyclicsinclude rings having from about 5 to about 9 carbon atoms in the ring.Alicyclic as used herein may optionally include further substitutentgroups.

The term “alkoxy,” as used herein, refers to a radical formed between analkyl group and an oxygen atom wherein the oxygen atom is used to attachthe alkoxy group to a parent molecule. Examples of alkoxy groupsinclude, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy,n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-henoxy andthe like. Alkoxy groups as used herein may optionally include furthersubstitutent groups.

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The terms “aryl” and “aromatic,” as used herein, refer to a mono- orpolycyclic carbocyclic ring system radicals having one or more aromaticrings. Examples of aryl groups include, but are not limited to, phenyl,naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferredaryl ring systems have from about 5 to about 20 carbon atoms in one ormore rings. Aryl groups as used herein may optionally include furthersubstitutent groups.

The terms “aralkyl” and “arylalkyl,” as used herein, refer to a radicalformed between an alkyl group and an aryl group wherein the alkyl groupis used to attach the aralkyl group to a parent molecule. Examplesinclude, but are not limited to, benzyl, phenethyl and the like. Aralkylgroups as used herein may optionally include further substitutent groupsattached to the alkyl, the aryl or both groups that form the radicalgroup.

The term “heterocyclic radical” as used herein, refers to a radicalmono-, or poly-cyclic ring system that includes at least one heteroatomand is unsaturated, partially saturated or fully saturated, therebyincluding heteroaryl groups. Heterocyclic is also meant to include fusedring systems wherein one or more of the fused rings contain at least oneheteroatom and the other rings can contain one or more heteroatoms oroptionally contain no heteroatoms. A heterocyclic group typicallyincludes at least one atom selected from sulfur, nitrogen or oxygen.Examples of heterocyclic groups include, [1,3]dioxolane, pyrrolidinyl,pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl,piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and thelike. Heterocyclic groups as used herein may optionally include furthersubstitutent groups.

The terms “heteroaryl,” and “heteroaromatic,” as used herein, refer to aradical comprising a mono- or poly-cyclic aromatic ring, ring system orfused ring system wherein at least one of the rings is aromatic andincludes one or more heteroatom. Heteroaryl is also meant to includefused ring systems including systems where one or more of the fusedrings contain no heteroatoms. Heteroaryl groups typically include onering atom selected from sulfur, nitrogen or oxygen. Examples ofheteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and thelike. Heteroaryl radicals can be attached to a parent molecule directlyor through a linking moiety such as an aliphatic group or hetero atom.Heteroaryl groups as used herein may optionally include furthersubstitutent groups.

The term “heteroarylalkyl,” as used herein, refers to a heteroaryl groupas previously defined having an alky radical that can attach theheteroarylalkyl group to a parent molecule. Examples include, but arenot limited to, pyridinylmethyl, pyrimidinylethyl, napthyridinylpropyland the like. Heteroarylalkyl groups as used herein may optionallyinclude further substitutent groups on one or both of the heteroaryl oralkyl portions.

The term “mono or poly cyclic structure” as used in the presentinvention includes all ring systems that are single or polycyclic havingrings that are fused or linked and is meant to be inclusive of singleand mixed ring systems individually selected from aliphatic, alicyclic,aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl,heteroaromatic, heteroarylalkyl. Such mono and poly cyclic structurescan contain rings that are uniform or have varying degrees of saturationincluding fully saturated, partially saturated or fully unsaturated.Each ring can comprise ring atoms selected from C, N, O and S to giverise to heterocyclic rings as well as rings comprising only C ring atomswhich can be present in a mixed motif such as for example benzimidazolewherein one ring has only carbon ring atoms and the fused ring has twonitrogen atoms. The mono or poly cyclic structures can be furthersubstituted with substituent groups such as for example phthalimidewhich has two ═O groups attached to one of the rings. In another aspect,mono or poly cyclic structures can be attached to a parent moleculedirectly through a ring atom, through a substituent group or abifunctional linking moiety.

The term “acyl,” as used herein, refers to a radical formed by removalof a hydroxyl group from an organic acid and has the general formula—C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examplesinclude aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls,aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substitutent groups.

The term “hydrocarbyl” includes groups comprising C, O and H. Includedare straight, branched and cyclic groups having any degree ofsaturation. Such hydrocarbyl groups can include one or more heteroatomsselected from N, O and S and can be further mono or poly substitutedwith one or more substituent groups.

The terms “substituent” and “substituent group,” as used herein, includegroups that are typically added to other groups or parent compounds toenhance desired properties or give desired effects. Substituent groupscan be protected or unprotected and can be added to one available siteor to many available sites in a parent compound. Substituent groups mayalso be further substituted with other substituent groups and may beattached directly or via a linking group such as an alkyl or hydrocarbylgroup to a parent compound. Such groups include without limitation,halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)R_(aa)), carboxyl(—C(O)O—R_(aa)), aliphatic groups, alicyclic groups, alkoxy, substitutedoxo (—O—R_(aa)), aryl, aralkyl, heterocyclic, heteroaryl,heteroarylalkyl, amino (—NR_(bb)R_(cc)), imino(═NR_(bb)), amido(—C(O)NR_(bb)R_(cc) or —N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂),cyano (—CN), carbamido (—OC(O)NR_(bb)R_(cc) or —N(R_(bb))C(O)OR_(aa)),ureido (—N(R_(bb))C(O)NR_(bb)R_(cc)), thioureido(—N(R_(bb))C(S)NR_(bb)R_(cc)), guanidinyl(—N(R_(bb))C(═NR_(bb))NR_(bb)R_(cc)), amidinyl(—C(═NR_(bb))NR_(bb)R_(cc) or —N(R_(bb))C(NR_(bb))R_(aa)), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)),sulfonamidyl (—S(O)₂NR_(bb)R_(cc) or —N(R_(bb))S(O)₂R_(bb)) andconjugate groups. Wherein each R_(aa), R_(bb) and R_(cc) is,independently, H, an optionally linked chemical functional group or afurther substituent group with a preferred list including withoutlimitation H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl,aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl.

B. CERTAIN OLIGOMERIC COMPOUNDS

In certain embodiments, it is desirable to chemically modify oligomericcompounds, compared to naturally occurring oligomers, such as DNA orRNA. Certain such modifications alter the activity of the oligomericcompound. Certain such chemical modifications can alter activity by, forexample: increasing affinity of an antisense compound for its targetnucleic acid, increasing its resistance to one or more nucleases, and/oraltering the pharmacokinetics or tissue distribution of the oligomericcompound. In certain instances, the use of chemistries that increase theaffinity of an oligomeric compound for its target can allow for the useof shorter oligomeric compounds.

1. Certain Monomers

In certain embodiment, oligomeric compounds comprise one or moremodified monomer. In certain such embodiments, oligomeric compoundscomprise one or more high affinity monomer. In certain embodiments, suchhigh-affinity monomer is selected from monomers (e.g., nucleosides andnucleotides) comprising 2′-modified sugars, including, but not limitedto: BNA's and monomers (e.g., nucleosides and nucleotides) with2′-substituents such as allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), or O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H or substituted or unsubstitutedC₁-C₁₀ alkyl.

In certain embodiments, the oligomeric compounds including, but nolimited to short antisense compounds of the present invention, compriseone or more high affinity monomers provided that the oligomeric compounddoes not comprise a nucleotide comprising a 2′-O(CH₂)_(n)H, wherein n isone to six.

In certain embodiments, the oligomeric compounds including, but nolimited to short antisense compounds of the present invention, compriseone or more high affinity monomer provided that the oligomeric compounddoes not comprise a nucleotide comprising a 2′-OCH₃ or a 2′-O(CH₂)₂OCH₃.

In certain embodiments, the oligomeric compounds including, but nolimited to short antisense compounds of the present invention, compriseone or more high affinity monomer provided that the oligomeric compounddoes not comprise a α-L-Methyleneoxy (4′-CH₂—O-2′) BNA.

In certain embodiments, the oligomeric compounds including, but nolimited to short antisense compounds of the present invention, compriseone or more high affinity monomer provided that the oligomeric compounddoes not comprise a β-D-Methyleneoxy (4′-CH₂—O-2′) BNA.

In certain embodiments, the oligomeric compounds including, but nolimited to short antisense compounds of the present invention, compriseone or more high affinity monomer provided that the oligomeric compounddoes not comprise a α-L-Methyleneoxy (4′-CH₂—O-2′) BNA orα-D-Methyleneoxy (4′-CH₂—O-2′) BNA.

a. Certain Nucleobases

The naturally occurring base portion of a nucleoside is typically aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. For those nucleosides thatinclude a pentofuranosyl sugar, a phosphate group can be linked to the2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides,those phosphate groups covalently link adjacent nucleosides to oneanother to form a linear polymeric compound. Within oligonucleotides,the phosphate groups are commonly referred to as forming theinternucleotide backbone of the oligonucleotide. The naturally occurringlinkage or backbone of RNA and of DNA is a 3′ to 5′ phosphodiesterlinkage.

In addition to “unmodified” or “natural” nucleobases such as the purinenucleobases adenine (A) and guanine (G), and the pyrimidine nucleobasesthymine (T), cytosine (C) and uracil (U), many modified nucleobases ornucleobase mimetics known to those skilled in the art are amenable withthe compounds described herein. In certain embodiments, a modifiednucleobase is a nucleobase that is fairly similar in structure to theparent nucleobase, such as for example a 7-deaza purine, a 5-methylcytosine, or a G-clamp. In certain embodiments, nucleobase mimeticinclude more complicated structures, such as for example a tricyclicphenoxazine nucleobase mimetic. Methods for preparation of the abovenoted modified nucleobases are well known to those skilled in the art.

b. Certain Sugars

Oligomeric compounds provided herein may comprise one or more monomer,including a nucleoside or nucleotide, having a modified sugar moiety.For example, the furanosyl sugar ring of a nucleoside can be modified ina number of ways including, but not limited to, addition of asubstituent group, bridging of two non-geminal ring atoms to form abicyclic nucleic acid (BNA).

In certain embodiments, oligomeric compounds comprise one or moremonomers that is a BNA. In certain such embodiments, BNA s include, butare not limited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B)β-D-Methyleneoxy (4′-CH₂—O-2′) BNA, (C) Ethyleneoxy (4′-(CH₂)₂—O-2′)BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA and (E) Oxyamino(4′-CH₂—N(R)—O-2′) BNA, as depicted in FIG. 1.

In certain embodiments, BNA compounds include, but are not limited to,compounds having at least one bridge between the 4′ and the 2′ positionof the sugar wherein each of the bridges independently comprises 1 orfrom 2 to 4 linked groups independently selected from —[C(R₁)(R₂)]_(n)—,—C(R₁)═C(R₂)—, —C(R₁)═N—, —C(═NR₁)—, —C(═O)—, —C(═S)—, —O—, —Si(R₁)₂—,—S(═O)_(x)— and —N(R₁)—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R₁ and R₂ is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycleradical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl,substituted C₁-C₁₂ aminoalkyl or a protecting group.

In one embodiment, each of the bridges of the BNA compounds is,independently, —[C(R₁)(R₂)]_(n)—[C(R₁)(R₂)]_(n)—O—, —C(R₁R₂)—N(R₁)—O— or—C(R₁R₂)—O—N(R₁)—. In another embodiment, each of said bridges is,independently,4′-CH₂-2′,4′-(CH₂)₂-2′,4′-(CH₂)₃-2′,4′-CH₂—O-2′,4′-(CH₂)₂—O-2′,4′-CH₂—O—N(R₁)-2′and 4′-CH₂—N(R₁)—O-2′- wherein each R₁ is, independently, H, aprotecting group or C₁-C₁₂ alkyl.

Certain BNA's have been prepared and disclosed in the patent literatureas well as in scientific literature (Singh et al., Chem. Commun., 1998,4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedtet al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar etal., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94/14226; WO2005/021570; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;Examples of issued US patents and published applications that discloseBNA s include, for example, U.S. Pat. Nos. 7,053,207; 6,268,490;6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-GrantPublication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841;2004-0143114; and 20030082807.

Also provided herein are BNAs in which the 2′-hydroxyl group of theribosyl sugar ring is linked to the 4′ carbon atom of the sugar ringthereby forming a methyleneoxy (4′-CH₂—O-2′) linkage to form thebicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens.Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 81-7; andOrum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S.Pat. Nos. 6,268,490 and 6,670,461). The linkage can be a methylene(—CH₂—) group bridging the 2′ oxygen atom and the 4′ carbon atom, forwhich the term methyleneoxy (4′-CH₂—O-2′) BNA is used for the bicyclicmoiety; in the case of an ethylene group in this position, the termethyleneoxy (4′-CH₂CH₂—O-2′) BNA is used (Singh et al., Chem. Commun.,1998, 4, 455-456: Morita et al., Bioorganic Medicinal Chemistry, 2003,11, 2211-2226). Methyleneoxy (4′-CH₂—O-2′) BNA and other bicyclic sugaranalogs display very high duplex thermal stabilities with complementaryDNA and RNA (Tm=+3 to +10° C.), stability towards 3′-exonucleolyticdegradation and good solubility properties. Potent and nontoxicantisense oligonucleotides comprising BNAs have been described(Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).

An isomer of methyleneoxy (4′-CH₂—O-2′) BNA that has also been discussedis alpha-L-methyleneoxy (4′-CH₂—O-2′) BNA which has been shown to havesuperior stability against a 3′-exonuclease. The alpha-L-methyleneoxy(4′-CH₂—O-2′) BNA's were incorporated into antisense gapmers andchimeras that showed potent antisense activity (Frieden et al., NucleicAcids Research, 2003, 21, 6365-6372).

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) BNAmonomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine anduracil, along with their oligomerization, and nucleic acid recognitionproperties have been described (Koshkin et al., Tetrahedron, 1998, 54,3607-3630). BNAs and preparation thereof are also described in WO98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA, phosphorothioate-methyleneoxy(4′-CH₂—O-2′) BNA and 2′-thio-BNAs, have also been prepared (Kumar etal., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation oflocked nucleoside analogs comprising oligodeoxyribonucleotide duplexesas substrates for nucleic acid polymerases has also been described(Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, anovel comformationally restricted high-affinity oligonucleotide analoghas been described in the art (Singh et al., J. Org. Chem., 1998, 63,10035-10039). In addition, 2′-Amino- and 2′-methylamino-BNA's have beenprepared and the thermal stability of their duplexes with complementaryRNA and DNA strands has been previously reported.

Modified sugar moieties are well known and can be used to alter,typically increase, the affinity of the antisense compound for itstarget and/or increase nuclease resistance. A representative list ofpreferred modified sugars includes but is not limited to bicyclicmodified sugars (BNA's), including methyleneoxy (4′-CH₂—O-2′) BNA andethyleneoxy (4′-(CH₂)₂—O-2′ bridge) BNA; substituted sugars, especially2′-substituted sugars having a 2′-F, 2′-OCH₃ or a 2′-O(CH₂)₂—OCH₃substituent group; and 4′-thio modified sugars. Sugars can also bereplaced with sugar mimetic groups among others. Methods for thepreparations of modified sugars are well known to those skilled in theart. Some representative patents and publications that teach thepreparation of such modified sugars include, but are not limited to,U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and 6,600,032;and WO 2005/121371.

In certain embodiments, BNA's include bicyclic nucleoside having theformula:

wherein:

Bx is a heterocyclic base moiety;

T₁ is H or a hydroxyl protecting group;

T₂ is H, a hydroxyl protecting group or a reactive phosphorus group;

Z is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl,substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl, substitutedacyl, or substituted amide.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with optionally protected substituent groupsindependently selected from halogen, oxo, hydroxyl, OJ₁, NJ₁J₂, SJ₁, N₃,OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ and J₃is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁.

In certain such embodiments, each of the substituted groups, is,independently, mono or poly substituted with substituent groupsindependently selected from halogen, oxo, hydroxyl, OJ₁, NJ₁J₂, SJ₁, N₃,OC(═X)J₁, and NJ₃C(═X)NJ₁J₂, wherein each J₁, J₂ and J₃ is,independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl and X is O orNJ₁.

In certain embodiments, the Z group is C₁-C₆ alkyl substituted with oneor more X^(x), wherein each X^(x) is independently OJ₁, NJ₁J₂, SJ₁, N₃,OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ or CN; wherein each J₁, J₂ and J₃is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁. In anotherembodiment, the Z group is C₁-C₆ alkyl substituted with one or moreX^(x), wherein each X^(x) is independently halo (e.g., fluoro),hydroxyl, alkoxy (e.g., CH₃O—), substituted alkoxy or azido.

In certain embodiments, the Z group is —CH₂X^(x), wherein X^(x) is OJ₁,NJ₃J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ or CN; wherein eachJ₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁.In another embodiment, the Z group is —CH₂X^(x), wherein X^(x) is halo(e.g., fluoro), hydroxyl, alkoxy (e.g., CH₃O—) or azido.

In certain such embodiments, the Z group is in the (R)-configuration:

In certain such embodiments, the Z group is in the (S)-configuration:

In certain embodiments, each T₁ and T₂ is a hydroxyl protecting group. Apreferred list of hydroxyl protecting groups includes benzyl, benzoyl,2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyl-diphenylsilyl,mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl)and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In certain embodiments, T₁is a hydroxyl protecting group selected from acetyl, benzyl,t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein amore preferred hydroxyl protecting group is T₁ is 4,4′-dimethoxytrityl.

In certain embodiments, T₂ is a reactive phosphorus group whereinpreferred reactive phosphorus groups include diisopropylcyanoethoxyphosphoramidite and H-phosphonate. In certain embodiments T₁ is4,4′-dimethoxytrityl and T₂ is diisopropylcyanoethoxy phosphoramidite.

In certain embodiments, oligomeric compounds have at least one monomerof the formula:

or of the formula:

or of the formula:

wherein

Bx is a heterocyclic base moiety;

T₃ is H, a hydroxyl protecting group, a linked conjugate group or aninternucleoside linking group attached to a nucleoside, a nucleotide, anoligonucleoside, an oligonucleotide, a monomeric subunit or anoligomeric compound;

T₄ is H, a hydroxyl protecting group, a linked conjugate group or aninternucleoside linking group attached to a nucleoside, a nucleotide, anoligonucleoside, an oligonucleotide, a monomeric subunit or anoligomeric compound;

wherein at least one of T₃ and T₄ is an internucleoside linking groupattached to a nucleoside, a nucleotide, an oligonucleoside, anoligonucleotide, a monomeric subunit or an oligomeric compound; and

Z is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl,substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl, substitutedacyl, or substituted amide.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with optionally protected substituent groupsindependently selected from halogen, oxo, hydroxyl, OJ₁, NJ₁J₂, SJ₁, N₃,OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ and J₃is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ₁, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, andNJ₃C(═X)NJ₁J₂, wherein each J₁, J₂ and J₃ is, independently, H or C₁-C₆alkyl, and X is O or NJ₁.

In certain such embodiments, at least one Z is C₁-C₆ alkyl orsubstituted C₁-C₆ alkyl. In certain embodiments, each Z is,independently, C₁-C₆ alkyl or substituted C₁-C₆ alkyl. In certainembodiments, at least one Z is C₁-C₆ alkyl. In certain embodiments, eachZ is, independently, C₁-C₆ alkyl. In certain embodiments, at least one Zis methyl. In certain embodiments, each Z is methyl. In certainembodiments, at least one Z is ethyl. In certain embodiments, each Z isethyl. In certain embodiments, at least one Z is substituted C₁-C₆alkyl. In certain embodiments, each Z is, independently, substitutedC₁-C₆ alkyl. In certain embodiments, at least one Z is substitutedmethyl. In certain embodiments, each Z is substituted methyl. In certainembodiments, at least one Z is substituted ethyl. In certainembodiments, each Z is substituted ethyl.

In certain embodiments, at least one substituent group is C₁-C₆ alkoxy(e.g., at least one Z is C₁-C₆ alkyl substituted with one or more C₁-C₆alkoxy). In another embodiment, each substituent group is,independently, C₁-C₆ alkoxy (e.g., each Z is, independently, C₁-C₆ alkylsubstituted with one or more C₁-C₆ alkoxy).

In certain embodiments, at least one C₁-C₆ alkoxy substituent group isCH₃O— (e.g., at least one Z is CH₃OCH₂—). In another embodiment, eachC₁-C₆ alkoxy substituent group is CH₃O— (e.g., each Z is CH₃OCH₂—).

In certain embodiments, at least one substituent group is halogen (e.g.,at least one Z is C₁-C₆ alkyl substituted with one or more halogen). Incertain embodiments, each substituent group is, independently, halogen(e.g., each Z is, independently, C₁-C₆ alkyl substituted with one ormore halogen). In certain embodiments, at least one halogen substituentgroup is fluoro (e.g., at least one Z is CH₂FCH₂—, CHF₂CH₂— or CF₃CH₂—).In certain embodiments, each halo substituent group is fluoro (e.g.,each Z is, independently, CH₂FCH₂—, CHF₂CH₂— or CF₃CH₂—).

In certain embodiments, at least one substituent group is hydroxyl(e.g., at least one Z is C₁-C₆ alkyl substituted with one or morehydroxyl). In certain embodiments, each substituent group is,independently, hydroxyl (e.g., each Z is, independently, C₁-C₆ alkylsubstituted with one or more hydroxyl). In certain embodiments, at leastone Z is HOCH₂—. In another embodiment, each Z is HOCH₂—.

In certain embodiments, at least one Z is CH₃—, CH₃CH₂—, CH₂OCH₃—, CH₂F—or HOCH₂—. In certain embodiments, each Z is, independently, CH₃—,CH₃CH₂—, CH₂OCH₃—, CH₂F— or HOCH₂—.

In certain embodiments, at least one Z group is C₁-C₆ alkyl substitutedwith one or more X^(x), wherein each X^(x) is, independently, OJ₁,NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ or CN; wherein eachJ₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁.In another embodiment, at least one Z group is C₁-C₆ alkyl substitutedwith one or more X^(x), wherein each X^(x) is, independently, halo(e.g., fluoro), hydroxyl, alkoxy (e.g., CH₃O—) or azido.

In certain embodiments, each Z group is, independently, C₁-C₆ alkylsubstituted with one or more X^(x), wherein each X^(x) is independentlyOJ₁, NJ₁J₂, SJ₁, N₃, OC(═X)₁₋₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ or CN;wherein each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and X isO, S or NJ₁. In another embodiment, each Z group is, independently,C₁-C₆ alkyl substituted with one or more X^(x), wherein each X^(x) isindependently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH₃O—) orazido.

In certain embodiments, at least one Z group is —CH₂X^(x), wherein X^(x)is OJ₁, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ or CN;wherein each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and X isO, S or NJ₁ In certain embodiments, at least one Z group is —CH₂X^(x),wherein X^(x) is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH₃O—) orazido.

In certain embodiments, each Z group is, independently, —CH₂X^(x),wherein each X^(x) is, independently, OJ₁, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ or CN; wherein each J₁, J₂ and J₃ is,independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁. In anotherembodiment, each Z group is, independently, —CH₂X^(x), wherein eachX^(x) is, independently, halo (e.g., fluoro), hydroxyl, alkoxy (e.g.,CH₃O—) or azido.

In certain embodiments, at least one Z is CH₃—. In another embodiment,each Z is, CH₃—.

In certain embodiments, the Z group of at least one monomer is in the(R)-configuration represented by the formula:

or the formula:

or the formula:

In certain embodiments, the Z group of each monomer of the formula is inthe (R)-configuration.

In certain embodiments, the Z group of at least one monomer is in the(S)— configuration represented by the formula:

or the formula:

or the formula:

In certain embodiments, the Z group of each monomer of the formula is inthe (S)-configuration.

In certain embodiments, T₃ is H or a hydroxyl protecting group. Incertain embodiments, T₄ is H or a hydroxyl protecting group. In afurther embodiment T₃ is an internucleoside linking group attached to anucleoside, a nucleotide or a monomeric subunit. In certain embodiments,T₄ is an internucleoside linking group attached to a nucleoside, anucleotide or a monomeric subunit. In certain embodiments, T₃ is aninternucleoside linking group attached to an oligonucleoside or anoligonucleotide. In certain embodiments, T₄ is an internucleosidelinking group attached to an oligonucleoside or an oligonucleotide. Incertain embodiments, T₃ is an internucleoside linking group attached toan oligomeric compound. In certain embodiments, T₄ is an internucleosidelinking group attached to an oligomeric compound. In certainembodiments, at least one of T₃ and T₄ comprises an internucleosidelinking group selected from phosphodiester or phosphorothioate.

In certain embodiments, oligomeric compounds have at least one region ofat least two contiguous monomers of the formula:

or of the formula:

or of the formula:

to

In certain embodiments, the oligomeric compound comprises at least tworegions of at least two contiguous monomers of the above formula. Incertain embodiments, the oligomeric compound comprises a gappedoligomeric compound. In certain embodiments, the oligomeric compoundcomprises at least one region of from about 8 to about 14 contiguousβ-D-2′-deoxyribofuranosyl nucleosides. In certain embodiments, theoligomeric compound comprises at least one region of from about 9 toabout 12 contiguous β-D-2′-deoxyribofuranosyl nucleosides.

In certain embodiments, monmers include sugar mimetics. In certain suchembodiments, a mimetic is used in place of the sugar orsugar-internucleoside linkage combination, and the nucleobase ismaintained for hybridization to a selected target. Representativeexamples of a sugar mimetics include, but are not limited to,cyclohexenyl or morpholino. Representative examples of a mimetic for asugar-internucleoside linkage combination include, but are not limitedto, peptide nucleic acids (PNA) and morpholino groups linked byuncharged achiral linkages. In some instances a mimetic is used in placeof the nucleobase. Representative nucleobase mimetics are well known inthe art and include, but are not limited to, tricyclic phenoxazineanalogs and universal bases (Berger et al., Nuc Acid Res. 2000,28:2911-14, incorporated herein by reference). Methods of synthesis ofsugar, nucleoside and nucleobase mimetics are well known to thoseskilled in the art.

3. Monomeric Linkages

Described herein are linking groups that link monomers (including, butnot limited to, modified unmodified nucleosides and nucleotides)together, thereby forming an oligomeric compound. The two main classesof linking groups are defined by the presence or absence of a phosphorusatom. Representative phosphorus containing linkages include, but are notlimited to, phosphodiesters (P═O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing linking groups include, but are not limitedto, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—), thiodiester (—O—C(O)—S—),thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)₂—O—); andN,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—). Oligomeric compoundshaving non-phosphorus linking groups are referred to asoligonucleosides. Modified linkages, compared to natural phosphodiesterlinkages, can be used to alter, typically increase, nuclease resistanceof the oligomeric compound. In certain embodiments, linkages having achiral atom can be prepared a racemic mixtures, as separate enantomers.Representative chiral linkages include, but are not limited to,alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing linkages are wellknown to those skilled in the art.

The oligomeric compounds described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), α or β such as for sugar anomers, or as(D) or (L) such as for amino acids et al. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

4. Oligomeric Compounds

In certain embodiments, provided herein are oligomeric compounds havingreactive phosphorus groups useful for forming linkages including forexample phosphodiester and phosphorothioate internucleoside linkages.Methods of preparation and/or purification of precursors or oligomericcompounds are not a limitation of the compositions or methods providedherein. Methods for synthesis and purification of oligomeric compoundsincluding DNA, RNA, oligonucleotides, oligonucleosides, and antisensecompounds are well known to those skilled in the art.

Generally, oligomeric compounds comprise a plurality of monomericsubunits linked together by linking groups. Nonlimiting examples ofoligomeric compounds include primers, probes, antisense compounds,antisense oligonucleotides, external guide sequence (EGS)oligonucleotides, alternate splicers, and siRNAs. As such, thesecompounds can be introduced in the form of single-stranded,double-stranded, circular, branched or hairpins and can containstructural elements such as internal or terminal bulges or loops.Oligomeric double-stranded compounds can be two strands hybridized toform double-stranded compounds or a single strand with sufficient selfcomplementarity to allow for hybridization and formation of a fully orpartially double-stranded compound.

In certain embodiments, the present invention provides chimericoligomeric compounds. In certain such embodiments, chimeric oligomericcompounds are chimeric oligonucleotides. In certain such embodiments,the chimeric oligonucleotides comprise differently modified nucleotides.In certain embodiments, chimeric oligonucleotides are mixed-backboneantisense oligonucleotides.

In general a chimeric oligomeric compound will have modified nucleosidesthat can be in isolated positions or grouped together in regions thatwill define a particular motif. Any combination of modifications and/ormimetic groups can comprise a chimeric oligomeric compound as describedherein.

In certain embodiments, chimeric oligomeric compounds typically compriseat least one region modified so as to confer increased resistance tonuclease degradation, increased cellular uptake, and/or increasedbinding affinity for the target nucleic acid. In certain embodiments, anadditional region of the oligomeric compound may serve as a substratefor enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase His a cellular endonuclease that cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofinhibition of gene expression. Consequently, comparable results canoften be obtained with shorter oligomeric compounds when chimeras areused, compared to for example phosphorothioate deoxyoligonucleotideshybridizing to the same target region. Cleavage of the RNA target can beroutinely detected by gel electrophoresis and, if necessary, associatednucleic acid hybridization techniques known in the art.

In certain embodiments, chimeric oligomeric compounds are gapmers. Incertain embodiments, chimeric compounds are short antisense compounds.In certain embodiments, short antisense compounds are gapmers. Incertain such embodiments, a mixed-backbone antisense oligomer has onetype of internucleotide linkages in one or both wings and a differenttype of internucleotide linkages in the gap. In certain suchembodiments, the mixed-backbone antisense oligonucleotide hasphosphodiester linkages in the wings and phosphorothioate linkages inthe gap. In certain embodiments in which the internucleotide linkages ina wing is different from the internucleotide linkages in the gap, theinternucleotide linkage bridging that wing and the gap is the same asthe internucleotide linkage in the wing. In certain embodiments in whichthe internucleotide linkages in a wing is different from theinternucleotide linkages in the gap, the internucleotide linkagebridging that wing and the gap is the same as the internucleotidelinkage in the gap.

C. CERTAIN SHORT ANTISENSE COMPOUNDS

Disclosed herein are short antisense compounds 8 to 16, preferably 9 to15, more preferably 9 to 14, more preferably 10 to 14 nucleotides inlength. In certain embodiments, short antisense compounds are 9 to 14nucleotides in length. In certain embodiments, short antisense compoundsare 10 to 14 nucleotides in length. In certain embodiments, such shortantisense compounds are short antisense oligonucleotides.

In certain embodiments, short antisense compounds comprise one or morechemical modifications. In certain such embodiments, short antisensecompounds comprise at least one modified nucleotide. In certainembodiments short antisense compounds comprise at least two or moremodified nucleotides. In certain embodiments, short antisense compoundscomprise at least one modified internucleotide linkage. In certainembodiments, short antisense compounds are mixed-backboneoligonucleotides. In certain embodiments, short antisense compounds arechimeric oligonucleotides. In certain embodiments, short antisenseoligonucleotides are uniformly modified. In certain embodiments, shortantisense oligonucleotides comprise modifications independently selectedat each nucleobase and at each linkage.

In certain embodiments, short antisense compounds are short gapmers. Incertain such embodiments, short gapmers comprise at least one highaffinity modification in one or more wings of the compound. In certainembodiments, short antisense compounds comprise 1 to 3 high-affinitymodifications in each wing. In certain embodiments, high affinitymodifications of the short antisense compounds allow for a targetaffinity similar to, or even greater than, the target affinity of longerantisense compounds. In certain embodiments, the high-affinity modifiednucleotides are sugar modified nucleotides. Such sugar modifiednucleotides include those comprising a bridge between the 4′ and 2′position of the sugar. Exemplary high affinity sugar modificationsinclude, but are not limited to, BNA s and other 2′-modifications suchas 2′-MOE. In an alternate embodiment of the invention, the highaffinity modification is not a 2′-O—(CH₂)_(n)H (n=1-6) sugar-modifiednucleotide. In an additional alternate embodiment, the high affinitymodified nucleotide is not a 2′-OCH₃ or a 2′-OCH₂CH₂OCH₃ nucleotide. Incertain embodiments, the high-affinity modified nucleotides confer aT_(m) of at least 1, at least 1.5, at least 2, at least 2.5, at least3.0, at least 3.5 or at least 4.0 degrees per nucleotide. Somehigh-affinity nucleotide modifications are known in the art to increasetoxicity. As shown herein, short antisense compounds having a limitednumber (generally 2 to 6) of high affinity modifications exhibit littleto no increase in toxicity but retain or increase affinity for thetarget RNA, while also significantly reducing expression of the RNAtarget. Short antisense compounds of the invention may optionallycomprise a conjugate group, such as, for example, cholesterol or C₁₆.

1. Certain Wings

In certain embodiments, the short antisense compounds comprise a 5′ wingand/or a 3′ wing. In such embodiments, the features of the 3′ wing andthe features of the 5′ wing are selected independently. Thus, in suchembodiments, the number of monomers in the 5′ wing and the number ofmonomers (length) in the 3′ wing may be the same or may be different;the modifications, if any, in the 5′ wing may be the same as themodifications, if any, in the 3′ wing or such modifications, if any, maybe different; and the monomeric linkages in the 5′ wing and themonomeric linkages in the 3′ wing may be the same or they may bedifferent.

In certain embodiments a wing comprises one, two or three monomers (i.e.has a length of 1, 2, or 3). In certain embodiments, the monomers of awing are modified. In certain such embodiments, the monomers of the wingare modified to increase affinity of the antisense compound for itstarget nucleic acid. In certain embodiments, the monomers of a wing arenucleosides or nucleotides. In certain such embodiments, the nucleosidesor nucleotides of the wing comprise a 2′ modification. In certain suchembodiments, the monomers (nucleosides or nucleotides) of the wing areBNA's. In certain such embodiments, the monomers of the wing areselected from α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, β-D-Methyleneoxy(4′-CH₂—O-2′) BNA, Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, Aminooxy(4′-CH₂—O—N(R)-2′) BNA and Oxyamino (4′-CH₂—N(R)—O-2′) BNA. In certainembodiments, the monomers of a wing comprise a substituent at the 2′position selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H or substituted or unsubstitutedC₁-C₁₀ alkyl. In certain embodiments, the monomers of a wing are 2′MOEnucleotides.

In certain embodiments, the monomeric linkages in a wing are naturallyoccurring internucleotide linkages. In certain embodiments, themonomeric linkages in a wing are non-naturally occurring internucleotideor internucleoside linkages. In certain such embodiments, the monomericlinkages in the wing are more resistant to one or more nucleases thannaturally occurring internucleotide linkages. In certain suchembodiments, the monomeric linkages in the wing are phosphorothioatelinkages (P═S). In certain embodiments where a wing has more than onemonomeric linkage, the monomeric linkages are the same as one another.In certain embodiments where a wing has more than one monomers linkage,the monomers linkages are different from each other.

One of ordinary skill in the art will recognize that the features andmodifications discussed above may be used in any combination to preparea wing. The table below provides non-limiting examples showing how onemight prepare a wing by selecting a certain number of monomers,monomeric modifications (if any), and monomeric linkages both within thewing.

Monomer type/ monomeric linkages Length modifications within wing 1 2′MOE None 1 BNA None 1 Methyleneoxy None BNA 1 ENA None 2 2′ MOE P═S 2BNA P═S 2 Methyleneoxy P═S BNA 2 ENA P═S 2 2′ MOE P═O 2 BNA P═O 2Methyleneoxy P═O BNA 2 ENA P═O 3 2′ MOE P═S 3 BNA P═S 3 Methyleneoxy P═SBNA 3 ENA P═S 3 2′ MOE P═O 3 BNA P═O 3 Methyleneoxy P═O BNA 3 ENA P═O

In certain embodiments in which a wing comprises two, three or fourmonomers, those two, three or four monomers all comprise the samemodifications, if any. In certain embodiments in which a wing comprisestwo, three or four monomers, one or more of those two, three or fournucleobases comprises one or more modifications that is different fromone or more of the modifications of one or more of the remainingmonomers.

2. Certain Gaps

In certain embodiments, the short antisense compounds comprise a gapbetween the 5′ wing and the 3′ wing. In certain embodiments the gapcomprises five, six, seven, eight, nine, ten, eleven, twelve, thirteen,or fourteen monomers. In certain embodiments, the monomers of the gapare unmodified deoxyribonucleotides. In certain embodiments, themonomers of the gap are unmodified ribonucleotides. In certainembodiments, gap modifications (if any) gap result in an antisensecompound that, when bound to its target nucleic acid, supports cleavageby an RNase, including, but not limited to, RNase H.

In certain embodiments, the monomeric linkages in the gap are naturallyoccurring internucleotide linkages. In certain embodiments, themonomeric linkages in the gap are non-naturally occurring linkages. Incertain such embodiments, the monomeric linkages in the gap are moreresistant to one or more nuclease than naturally occurringinternucleotide linkages. In certain such embodiments, the monomericlinkages in the gap are phosphorothioate linkages (P═S). In certainembodiments, the monomeric linkages in the gap are all the same as oneanother. In certain embodiments, the monomeric linkages within the gapare not all the same.

One of ordinary skill in the art will recognize that the features andmodifications discussed above may be used in any combination to preparea gap. The table below provides non-limiting examples showing how onemight prepare a gap by selecting a certain number of monomers, monomericmodifications (if any), and monomeric linkages within the gap region.

Monomer type/ Monomeric linkages Length modifications within gap 5 DNAP═S 6 DNA P═S 7 DNA P═S 8 DNA P═S 9 DNA P═S 10 DNA P═S 11 DNA P═S 12 DNAP═S 13 DNA P═S 14 DNA P═S 6 DNA P═O 7 DNA P═O 8 DNA P═O 9 DNA P═O 10 DNAP═O 11 DNA P═O 12 DNA P═O 8 RNA P═S 9 RNA P═S 10 RNA P═S 11 RNA P═S 12RNA P═S

3. Certain Gapped Antisense Oligomeric Compounds

One of ordinary skill in the art will recognize that the wings and thegaps discussed above may be selected and then combined in a variety ofcombinations to generate gapped oligomeric compounds, including, but notlimited to, gapped antisense oligomeric compounds, and gapped antisenseoligonucleotides. The features (length, modifications, linkages) of the5′ wing and the 3′ wing may be selected independently of one another.The features of the gap include at least one difference in modificationcompared to the features of the 5′ wing and at least one differencecompared to the features of the 3′ wing (i.e., there must be at leastone difference in modification between neighboring regions todistinguish those neighboring regions from one another). The features ofthe gap may otherwise be selected independently.

In certain embodiments, the monomeric linkages within a wing and themonomeric linkages within the gap are the same. In certain embodiments,the monomeric linkages within a wing and the monomeric linkages withinthe gap are different. In certain such embodiments, the monomericlinkage bridging the wing and the gap are the same as the monomericlinkages in the wing. In certain embodiments, the monomeric linkagebridging the wing and the gap are the same as the monomeric linkages inthe gap. In certain embodiments, short antisense compounds have uniformlinkages throughout the compound. In certain such embodiments, all ofthe linkages are phosphorothioate (P═S) linkages.

One of ordinary skill in the art will recognize that the 3′ wings, 5′wings, gaps, and linkages discussed above may be used in any combinationto prepare a gapmer. The table below provides non-limiting examplesshowing how one might prepare a gapmer by selecting a certain 5′ wing, agap, a 3′ wing and certain linkages bridging the gap and each wing.

5′ 3′ 5′ Wing Bridge Gap Bridge 3′ Wing Length Monomer Link Link LengthMonomer Link Link Length Monomer Link 2 MOE P═S P═S 6 DNA P═S P═S 2 MOEP═S 2 BNA P═S P═O 8 DNA P═O P═S 3 BNA P═S 1 MOE None P═S 10 DNA P═S P═S1 MOE P═S 2 MOE P═S P═S 8 RNA P═S P═S 2 MOE P═S 3 Methyleneoxy P═S P═S 8RNA P═S P═S 3 MOE P═S BNA 3 DNA P═O P═O 10 RNA P═S P═O 3 2′OH P═O 2 2-FP═S P═S 5 RNA P═S P═S 2 2′-F P═S 1 MOE P═O P═S 5 DNA P═O P═S 4 MOE P═S

In certain embodiments, the oligomeric compounds disclosed herein maycomprise from about 8 to about 16, preferably 9 to 15, more preferably 9to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16linked monomers). One of ordinary skill in the art will appreciate thatthis comprehends antisense compounds of 8, 9, 10, 11, 12, 13, 14, 15 or16 nucleobases. In certain embodiments, oligomeric compounds areantisense compounds.

In certain embodiments, short antisense compounds are 8 nucleobases inlength.

In certain embodiments, short antisense compounds are 9 nucleobases inlength.

In certain embodiments, short antisense compounds are 10 nucleobases inlength.

In certain embodiments, short antisense compounds are 11 nucleobases inlength.

In certain embodiments, short antisense compounds are 12 nucleobases inlength.

In certain embodiments, short antisense compounds are 13 nucleobases inlength.

In certain embodiments, short antisense compounds are 14 nucleobases inlength.

In certain embodiments, short antisense compounds are 15 nucleobases inlength.

In certain embodiments, short antisense compounds are 16 nucleobases inlength.

In certain embodiments, short antisense compounds are 8 monomers inlength. In certain embodiments, short antisense compounds are 9 monomersin length. In certain embodiments, short antisense compounds are 10monomers in length. In certain embodiments, short antisense compoundsare 11 monomers in length. In certain embodiments, short antisensecompounds are monomers in length. In certain embodiments, shortantisense compounds are 13 monomers in length. In certain embodiments,short antisense compounds are 14 monomers in length. In certainembodiments, short antisense compounds are 15 monomers in length. Incertain embodiments, short antisense compounds are 16 monomers inlength. In certain embodiments, short antisense compounds comprise 9 to15 monomers. In certain embodiments, short antisense compounds comprise10 to 15 monomers. In certain embodiments, short antisense compoundscomprise 12 to 14 monomers. In certain embodiments, short antisensecompounds comprise 12 to 14 nucleotides or nucleosides.

One having skill in the art and informed by the short antisensecompounds illustrated herein will be able, without undueexperimentation, to identify further short antisense compounds.

In certain embodiments, short antisense compounds comprise a gap flankedby more than one wing on either or both sides. Thus, in certainembodiments, a short antisense compound comprises two or more 5′ wingsand two or more 3′ wings. In certain embodiments, a short antisensecompound comprises one 5′ wing and two or more 3′ wings. In certainembodiments, a short antisense compound comprises one 3′ wing and two ormore 5′ wings. Certain such embodiments comprise, for example, thefollowing regions: a first 5′ wing—a bridge—a second 5′ wing—a bridge—agap—a bridge—a second 3′ wing—a bridge—a first 3′wing. In suchembodiments, each region has at least one difference in modificationwhen compared to its neighboring region. Thus, in such embodiments, thesecond 5′ wing and the second 3′ wing each independently comprises oneor more differences in modification compared to the gap and compared tothe first 5′ wing and the first 3′ wing. In such embodiments, themodifications of the first 3′ wing and first 5′ wing may either or bothbe the same or different from the modifications of the gap, if any.

4. Certain Conjugate Groups

In one aspect, oligomeric compounds are modified by covalent attachmentof one or more conjugate groups. In general, conjugate groups modify oneor more properties of the attached oligomeric compound including but notlimited to pharmacodynamic, pharmacokinetic, binding, absorption,cellular distribution, cellular uptake, charge and clearance. Conjugategroups are routinely used in the chemical arts and are linked directlyor via an optional linking moiety or linking group to a parent compoundsuch as an oligomeric compound. A preferred list of conjugate groupsincludes without limitation, intercalators, reporter molecules,polyamines, polyamides, polyethylene glycols, thioethers, polyethers,cholesterols, thiocholesterols, cholic acid moieties, folate, lipids,phospholipids, biotin, phenazine, phenanthridine, anthraquinone,adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.

Preferred conjugate groups amenable to the present invention includelipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al.,Bioorg. Med. Chem. Lett., 1994, 4, 1053); a thioether, e.g.,hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); athiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259,327; Svinarchuk et al., Biochimie, 1993, 75, 49); a phospholipid, e.g.,di-hexadecyl-rac-glycerol ortriethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl.Acids Res., 1990, 18, 3777); a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); apalmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264,229); or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety(Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Linking groups or bifunctional linking moieties such as those known inthe art are amenable to the compounds provided herein. Linking groupsare useful for attachment of chemical functional groups, conjugategroups, reporter groups and other groups to selective sites in a parentcompound such as for example an oligomeric compound. In general abifunctional linking moiety comprises a hydrocarbyl moiety having twofunctional groups. One of the functional groups is selected to bind to aparent molecule or compound of interest and the other is selected tobind essentially any selected group such as chemical functional group ora conjugate group. In some embodiments, the linker comprises a chainstructure or an oligomer of repeating units such as ethylene glycol oramino acid units. Examples of functional groups that are routinely usedin a bifunctional linking moiety include, but are not limited to,electrophiles for reacting with nucleophilic groups and nucleophiles forreacting with electrophilic groups. In some embodiments, bifunctionallinking moieties include amino, hydroxyl, carboxylic acid, thiol,unsaturations (e.g., double or triple bonds), and the like. Somenonlimiting examples of bifunctional linking moieties include8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

5. Synthesis, Purification and Analysis

Oligomerization of modified and unmodified nucleosides and nucleotidescan be routinely performed according to literature procedures for DNA(Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), HumanaPress) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al.,Applications of Chemically synthesized RNA in RNA: Protein Interactions,Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57,5707-5713).

Oligomeric compounds provided herein can be conveniently and routinelymade through the well-known technique of solid phase synthesis.Equipment for such synthesis is sold by several vendors including, forexample, Applied Biosystems (Foster City, Calif.). Any other means forsuch synthesis known in the art may additionally or alternatively beemployed. It is well known to use similar techniques to prepareoligonucleotides such as the phosphorothioates and alkylatedderivatives. The invention is not limited by the method of antisensecompound synthesis.

Methods of purification and analysis of oligomeric compounds are knownto those skilled in the art. Analysis methods include capillaryelectrophoresis (CE) and electrospray-mass spectroscopy. Such synthesisand analysis methods can be performed in multi-well plates. The methodof the invention is not limited by the method of oligomer purification.

D. ANTISENSE

Antisense mechanisms are all those involving the hybridization of acompound with target nucleic acid, wherein the outcome or effect of thehybridization is either target degradation or target occupancy withconcomitant stalling of the cellular machinery involving, for example,transcription or splicing.

One type of antisense mechanism involving target degradation includes anRNase H. RNase His a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. It is known in the art that single-strandedantisense compounds which are “DNA-like” elicit RNAse H activity inmammalian cells. Activation of RNase H, therefore, results in cleavageof the RNA target, thereby greatly enhancing the efficiency of DNA-likeoligonucleotide-mediated inhibition of gene expression.

In certain embodiments, chemically-modified antisense compounds have ahigher affinity for target RNAs than does non-modified DNA. In certainsuch embodiments, that higher affinity in turn provides increasedpotency allowing for the administration of lower doses of suchcompounds, reduced potential for toxicity and improvement in therapeuticindex and decreased overall cost of therapy.

The present disclosure demonstrates that the incorporation ofchemically-modified high-affinity nucleotides and nucleosides intoantisense compounds allows for the design of short antisense compounds8-16 nucleobases in length useful for the reduction of target RNAsand/or target proteins in cells, tissues, and animals, including, butnot limited to, humans with increased potency and improved therapeuticindex. Thus, in certain embodiments, provided herein are short antisensecompounds comprising high-affinity nucleotide modifications useful forreducing a target RNA in vivo. Certain such short antisense compoundsare effective at lower doses than previously described antisensecompounds, allowing for a reduction in toxicity and cost of treatment.In addition, certain short antisense compounds have greater potentialfor oral dosing.

To address the need for more potent antisense compounds, provided hereinare short antisense compounds (8-16, preferably 9 to 15, more preferably9 to 14, more preferably 10 to 14 nucleotides in length) with increasedactivity in vivo relative to longer compounds. Certain short antisensecompounds are gapmer compounds comprising high-affinitychemically-modified nucleotides on the 3′ and 5′ ends (wings) of thecompound. In certain embodiments, the addition of high-affinity modifiednucleotides allows antisense compounds to be active against, andspecific for, their intended target RNA in vivo despite being shorter inlength. Contemplated herein are short antisense compounds wherein eachof the wings independently comprises 1 to 3 high-affinity modifiednucleotides. In certain embodiments, the high-affinity modifications aresugar modifications. High-affinity modified nucleotides include, but arenot limited to, BNA s or other 2′-modified nucleotides, such as 2′-MOEnucleotides. Also contemplated are short antisense compounds having atleast one modified internucleotide linkage, such as a phosphorothioateinternucleotide linkage. In certain embodiments, the short antisensecompounds of the present invention can have all phosphorothioateinternucleoside linkages. The short antisense compounds optionallycomprise a conjugate group. As shown herein, short antisense compoundshave greater affinity for target RNA than they have for DNA and aresignificantly more potent in vivo as shown by reduction of target mRNAas well as by amelioration of a variety of disease indications.

As used herein, an RNA which is involved in regulating glucosemetabolism or clearance, lipid metabolism, cholesterol metabolism orinsulin metabolism is any RNA involved in the biochemical pathways thatregulate these processes. Such RNAs are well known in the art. Examplesof target genes include, but are not limited to, ApoB-100 (also known asAPOB; Ag(x) antigen; apoB-48; apolipoprotein B; apolipoprotein B-100;apolipoprotein B-48) and GCGR (also known as glucagon receptor; GR),CRP, DGAT2, GCCR, PCSK9, PTEN, PTP1B, SGLT2, and SOD1.

1. Modulation of Target Expression

In certain embodiments, a target is identified and antisenseoligonucleotides are designed to modulate that target or its expression.In certain embodiments, designing an oligomeric compound to a targetnucleic acid molecule can be a multistep process. Typically the processbegins with the identification of a target protein, the activity ofwhich is to be modulated, and then identifying the nucleic acid theexpression of which yields the target protein. In certain embodiments,designing of an antisense compound results in an antisense compound thatis hybridizable to the targeted nucleic acid molecule. In certainembodiments, the antisense compound is an antisense oligonucleotide orantisense oligonucleoside. In certain embodiments, an antisense compoundand a target nucleic acid are complementary to one another. In certainsuch embodiments, an antisense compound is perfectly complementary to atarget nucleic acid. In certain embodiments, an antisense compoundincludes one mismatch. In certain embodiments, an antisense compoundincludes two mismatches. In certain embodiments, an antisense compoundincludes three or more mismatches.

Modulation of expression of a target nucleic acid can be achievedthrough alteration of any number of nucleic acid functions. In certainembodiments, the functions of RNA to be modulated include, but are notlimited to, translocation functions, which include, but are not limitedto, translocation of the RNA to a site of protein translation,translocation of the RNA to sites within the cell which are distant fromthe site of RNA synthesis, and translation of protein from the RNA. RNAprocessing functions that can be modulated include, but are not limitedto, splicing of the RNA to yield one or more RNA species, capping of theRNA, 3′ maturation of the RNA and catalytic activity or complexformation involving the RNA which may be engaged in or facilitated bythe RNA. Modulation of expression can result in the increased level ofone or more nucleic acid species or the decreased level of one or morenucleic acid species, either temporally or by net steady state level.Thus, in one embodiment modulation of expression can mean increase ordecrease in target RNA or protein levels. In another embodimentmodulation of expression can mean an increase or decrease of one or moreRNA splice products, or a change in the ratio of two or more spliceproducts.

In certain embodiments, expression of a target gene is modulated usingan oligomeric compound comprising from about 8 to about 16, preferably 9to 15, more preferably 9 to 14, more preferably 10 to 14 monomers (i.e.from about 8 to about 16 linked monomers). One of ordinary skill in theart will appreciate that this comprehends methods of modulatingexpression of a target gene using one or more antisense compounds of 8,9, 10, 11, 12, 13, 14, 15 or 16 nucleobases.

In certain embodiments, methods of modulating a target gene comprisesuse of a short antisense compound that is 8 nucleobases in length. Incertain embodiments, methods of modulating a target gene comprises useof a short antisense compound that is 9 nucleobases in length. Incertain embodiments, methods of modulating a target gene comprises useof a short antisense compound that is 8 nucleobases in length. Incertain embodiments, methods of modulating a target gene comprises useof a short antisense compound that is 10 nucleobases in length. Incertain embodiments, methods of modulating a target gene comprises useof a short antisense compound that is 10 nucleobases in length. Incertain embodiments, methods of modulating a target gene comprises useof a short antisense compound that is 11 nucleobases in length. Incertain embodiments, methods of modulating a target gene comprises useof a short antisense compound that is 12 nucleobases in length. Incertain embodiments, methods of modulating a target gene comprises useof a short antisense compound that is 13 nucleobases in length. Incertain embodiments, methods of modulating a target gene comprises useof a short antisense compound that is 14 nucleobases in length. Incertain embodiments, methods of modulating a target gene comprises useof a short antisense compound that is 15 nucleobases in length. Incertain embodiments, methods of modulating a target gene comprises useof a short antisense compound that is 16 nucleobases in length.

In certain embodiments, methods of modulating expression of a targetgene comprises use of a short antisense compound comprising 9 to 15monomers. In certain embodiments, methods of modulating expression of atarget gene comprises use of a short antisense compound comprising 10 to15 monomers. In certain embodiments, methods of modulating expression ofa target gene comprises use of a short antisense compound comprising 12to 14 monomers. In certain embodiments, methods of modulating expressionof a target gene comprises use of a short antisense compound comprising12 or 14 nucleotides or nucleosides.

2. Hybridization

In certain embodiments, antisense compounds specifically hybridize whenthere is a sufficient degree of complementarity to avoid non-specificbinding of the antisense compound to non-target nucleic acid sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

As used herein, “stringent hybridization conditions” or “stringentconditions” refers to conditions under which an antisense compound willhybridize to its target sequence, but to a minimal number of othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances, and “stringent conditions” underwhich antisense compounds hybridize to a target sequence are determinedby the nature and composition of the antisense compounds and the assaysin which they are being investigated.

3. Complementarity

It is understood in the art that incorporation of nucleotide affinitymodifications may allow for a greater number of mismatches compared toan unmodified compound. Similarly, certain oligonucleotide sequences maybe more tolerant to mismatches than other oligonucleotide sequences. Oneof ordinary skill in the art is capable of determining an appropriatenumber of mismatches between oligonucleotides, between anoligonucleotide and a target nucleic acid, such as by determiningmelting temperature (T_(m)). T_(m) or T_(m) can be calculated bytechniques that are familiar to one of ordinary skill in the art. Forexample, techniques described in Freier et al. (Nucleic Acids Research,1997, 25, 22: 4429-4443) allow one of ordinary skill in the art toevaluate nucleotide modifications for their ability to increase themelting temperature of an RNA:DNA duplex.

4. Identity

Antisense compounds, or a portion thereof, may have a defined percentidentity to a SEQ ID NO, or a compound having a specific Isis number. Asused herein, a sequence is identical to the sequence disclosed herein ifit has the same nucleobase pairing ability. For example, an RNA whichcontains uracil in place of thymidine in the disclosed sequences of thecompounds described herein would be considered identical as they bothpair with adenine. This identity may be over the entire length of theoligomeric compound, or in a portion of the antisense compound (e.g.,nucleobases 1-20 of a 27-mer may be compared to a 20-mer to determinepercent identity of the oligomeric compound to the SEQ ID NO. It isunderstood by those skilled in the art that an antisense compound neednot have an identical sequence to those described herein to functionsimilarly to the antisense compound described herein. Shortened versionsof antisense compounds taught herein, or non-identical versions of theantisense compounds taught herein, are also provided herein.Non-identical versions are those wherein each base does not have thesame pairing activity as the antisense compounds disclosed herein. Basesdo not have the same pairing activity by being shorter or having atleast one abasic site. Alternatively, a non-identical version caninclude at least one base replaced with a different base with differentpairing activity (e.g., G can be replaced by C, A, or T). Percentidentity is calculated according to the number of bases that haveidentical base pairing corresponding to the SEQ ID NO or antisensecompound to which it is being compared. The non-identical bases may beadjacent to each other, dispersed through out the oligonucleotide, orboth.

For example, a 16-mer having the same sequence as nucleobases 2-17 of a20-mer is 80% identical to the 20-mer. Alternatively, a 20-mercontaining four nucleobases not identical to the 20-mer is also 80%identical to the 20-mer. A 14-mer having the same sequence asnucleobases 1-14 of an 18-mer is 78% identical to the 18-mer. Suchcalculations are well within the ability of those skilled in the art.

The percent identity is based on the percent of nucleobases in theoriginal sequence present in a portion of the modified sequence.Therefore, a 30 nucleobase antisense compound comprising the fullsequence of the complement of a 20 nucleobase active target segmentwould have a portion of 100% identity with the complement of the 20nucleobase active target segment, while further comprising an additional10 nucleobase portion. In the context of the instant description, thecomplement of an active target segment may constitute a single portion.In preferred embodiments, the oligonucleotides provided herein are atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% identical to at least aportion of the complement of the active target segments presentedherein.

E. TARGET NUCLEIC ACIDS, REGIONS AND SEGMENTS

In certain embodiments, short antisense compounds may be designed totarget any target nucleic acid. In certain embodiments, the targetnucleic acid encodes a target that is clinically relevant. In suchembodiments, modulation of the target nucleic acid results in clinicalbenefit. Certain target nucleic acids include, but are not limited to,the target nucleic acids illustrated in Table 1.

In certain embodiments, a target nucleic acid is a nucleic acid moleculeencoding ApoB. Nucleic acid molecules that encode ApoB include, withoutlimitation, SEQ ID NO: 1 and SEQ ID NO: 2.

In certain embodiments, a target nucleic acid is a nucleic acid moleculeencoding SGLT2. Nucleic acid molecules that encode SGLT2 include,without limitation, SEQ ID NO: 3.

In certain embodiments, a target nucleic acid is a nucleic acid moleculeencoding PCSK9. Nucleic acid molecules that encode PCSK9 include,without limitation, SEQ ID NO: 4.

In certain embodiments, a target nucleic acid is a nucleic acid moleculeencoding SOD 1. Nucleic acid molecules that encode SOD1 include, withoutlimitation, SEQ ID NO: 5.

In certain embodiments, a target nucleic acid is a nucleic acid moleculeencoding CRP. Nucleic acid molecules that encode CRP include, withoutlimitation, SEQ ID NO: 6.

In certain embodiments, a target nucleic acid is a nucleic acid moleculeencoding GCCR. Nucleic acid molecules that encode GCCR include, withoutlimitation, SEQ ID NO: 7 and SEQ ID NO: 8.

In certain embodiments, a target nucleic acid is a nucleic acid moleculeencoding GCGR. Nucleic acid molecules that encode GCGR include, withoutlimitation, SEQ ID NO: 9.

In certain embodiments, a target nucleic acid is a nucleic acid moleculeencoding DGAT2. Nucleic acid molecules that encode DGAT2 include,without limitation, SEQ ID NO: 10.

In certain embodiments, a target nucleic acid is a nucleic acid moleculeencoding PTP1B. Nucleic acid molecules that encode PTP1B include,without limitation, SEQ ID NO: 11 and SEQ ID NO: 12.

In certain embodiments, a target nucleic acid is a nucleic acid moleculeencoding PTEN. Nucleic acid molecules that encode PTEN include, withoutlimitation, SEQ ID NO: 14 or SEQ ID NO: 15.

TABLE 1 Certain Target Nucleic Acids GENBANK ® SEQ ID Target SpeciesAccession Number NO ApoB Human NM_000384.1 1 ApoB Mouse XM_137955.5 2SGLT2 Human NM_003041.1 3 PCSK9 Human NM_174936.2 4 SOD1 Human X02317.15 CRP Human NM_000567.1 6 GCCR Mouse BC031885.1 7 GCCR Human Nucleotides1 to 10600 8 of AC012634 GCGR Human NM_000160.1 9 DGAT2 HumanNM_032564.2 10 PTP1B Human NM_002827.2 11 PTP1B Human Nucleotides1417800 12 to 1425600 of NT_011362.9 PTEN Mouse U92437.1 13 PTEN HumanNM_000314.4 14 PTEN Human Nucleotides 8063255 to 15 8167140 ofNT_033890.3

The targeting process usually includes determination of at least onetarget region, segment, or site within the target nucleic acid for theantisense interaction to occur such that the desired effect will result.

In certain embodiments, the 5′-most nucleotide of a target region is the5′ target site of a short antisense compound and the 3′-most nucleotideof a target region is the 3′ target site of the same short antisensecompound. In certain embodiments, the 5′-most nucleotide of a targetregion is the 5′ target site of a short antisense compound and the3′-most nucleotide of a target region is the 3′ target site of adifferent short antisense compound. In certain embodiments, a targetregion comprises a nucleotide sequence within 10, 15, or 20 nucleotidesof a 5′ target site or a 3′ target site.

In certain embodiments, a target region is a structurally defined regionof the nucleic acid. For example, in certain such embodiments, a targetregion may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region.

The locations on the target nucleic acid defined by having one or moreactive short antisense compounds targeted thereto are referred to as“active target segments.” In certain embodiments, the target nucleicacid having one or more active short antisense compounds targetedthereto is a target RNA. When an active target segment is defined bymultiple short antisense compounds, the compounds are preferablyseparated by no more than about 10 nucleotides on the target sequence,more preferably no more than about 5 nucleotides on the target sequence,even more preferably the short antisense compounds are contiguous, mostpreferably the short antisense compounds are overlapping. There may besubstantial variation in activity (e.g., as defined by percentinhibition) of the short antisense compounds within an active targetsegment. Active short antisense compounds are those that modulate theexpression of their target nucleic acid, including but not limited to atarget RNA. Active short antisense compounds inhibit expression of theirtarget RNA at least 10%, preferably 20%. In a preferred embodiment, atleast about 50%, preferably about 70% of the short antisense compoundstargeted to the active target segment modulate expression of theirtarget RNA at least 40%. In a more preferred embodiment, the level ofinhibition required to define an active short antisense compound isdefined based on the results from the screen used to define the activetarget segments.

A suitable target segment is at least about an 8-nucleobase portion of atarget region to which an active short antisense compound is targeted.Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative target segments (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA comprises about 8 to about 16 nucleobases). Targetsegments are also represented by DNA or RNA sequences that comprise atleast the 8 consecutive nucleobases from the 3′-terminus of one of theillustrative target segments (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelydownstream of the 3′-terminus of the target segment and continuing untilthe DNA or RNA comprises about 8 to about 16 nucleobases). It is alsounderstood that antisense target segments may be represented by DNA orRNA sequences that comprise at least 8 consecutive nucleobases from aninternal portion of the sequence of an illustrative target segment, andmay extend in either or both directions until the short antisensecompound comprises about 8 to about 16 nucleobases. One having skill inthe art armed with the target segments illustrated herein will be able,without undue experimentation, to identify further target segments.

Once one or more target regions, segments or sites have been identified,short antisense compounds are chosen which are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, to give the desired effect.

The short antisense compounds may also be targeted to regions of thetarget nucleobase sequence comprising any consecutive nucleobases 8 to16 nucleobases in length along the target nucleic acid molecule.

Target segments 8-16 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative target segments are considered to be suitable for targetingas well. Thus, the short antisense compounds may also encompass 8-16nucleobases within those segments identified herein as beginning at aparticular 5′ target site. Any segment of 8, 9, 10, 11, or morepreferably 12, 13, 14, 15 or 16 contiguous nucleobases in a 50,preferably 25, more preferably 16 nucleobase perimeter around theseregions are also considered to be suitable for targeting.

In a further embodiment, the “suitable target segments” identifiedherein may be employed in a screen for additional short antisensecompounds that modulate the expression of a target nucleic acid.“Modulators” are those compounds that decrease or increase theexpression of a target nucleic acid and which comprise at least an8-nucleobase portion which is complementary to a target segment. Thescreening method comprises the steps of contacting a target segment of anucleic acid with one or more candidate modulators, and selecting forone or more candidate modulators which decrease or increase theexpression of a target nucleic acid. Once it is shown that the candidatemodulator or modulators are capable of modulating (e.g. eitherdecreasing or increasing) the expression of a target nucleic acid, themodulator may then be employed in further investigative studies of thefunction of the target, or for use as a research, diagnostic, ortherapeutic agent in accordance with the present invention.

For all short antisense compounds discussed herein, sequence, monomer,monomeric modification, and monomeric linkage may each be selectedindependently. In certain embodiments, short antisense compounds aredescribed by a motif. In such embodiments, any motif may be used withany sequence, whether or not the sequence and/or the motif isspecifically disclosed herein. In certain embodiments, short antisensecompounds comprise modifications that are not amenable to description bymotif (for example, short antisense compounds comprising severaldifferent modifications and/or linkages at various positions throughoutthe compound). Such combinations may be incorporated for any sequence,whether or not it is disclosed herein. The sequence listing accompanyingthis filing provides certain nucleic acid sequences independent ofchemical modification. Though that listing identifies each sequence aseither “RNA” or “DNA” as required, in reality, those sequences may bemodified with any combination of chemical modifications and/or motifs.

In certain embodiments, short antisense compounds comprise at least onehigh-affinity modified monomer. In certain embodiments, provided areshort antisense compounds targeted to nucleic acid molecules encodingtargets including, but not limited to, ApoB-100 (also known as APOB;Ag(x) antigen; apoB-48; apolipoprotein B; apolipoprotein B-100;apolipoprotein B-48), GCGR (also known as glucagon receptor; GR), CRP,DGAT2, GCCR, PCSK9, PTEN, PTP1B, SGLT2, and SOD1. In certain suchembodiments, such short antisense compounds are targeted to a nucleicacid molecule encoding any of those targets.

F. CERTAIN TARGETS

In certain embodiments, short antisense compounds may be designed tomodulate any target. In certain embodiments, the target is clinicallyrelevant. In such embodiments, modulation of the target results inclinical benefit. Certain targets are preferentially expressed in thekidney. Certain targets are preferentially expressed in the liver.Certain targets are associated with a metabolic disorder. Certaintargets are associated to a cardiovascular disorder. In certainembodiments, a target is selected from: ApoB, SGLT2, PCSK9, SOD1, CRP,GCCR, GCGR, DGAT2, PTP1B, and PTEN. In certain embodiments, a target isselected from: ApoB, SGLT2, PCSK9, SOD1, CRP, GCCR, GCGR, DGAT2, andPTP1B. In certain embodiments, a target is any protein other than SGLT2.

In certain embodiments, short antisense compounds exhibit liver andkidney-specific target RNA reduction in vivo. Such property rendersthose short antisense compounds particularly useful for inhibition ofmany target RNAs involved in metabolic and cardiovascular diseases.Thus, provided herein are methods of treating cardiovascular ormetabolic disorders by contacting said kidney or liver tissues withshort antisense compounds targeted to RNAs associated with saiddisorders. Thus, also provided are methods for ameliorating any of avariety of metabolic or cardiovascular disease indications with theshort antisense compounds of the present invention.

1. ApoB

ApoB (also known as apolipoprotein B-100; ApoB-100, apolipoprotein B-48;ApoB-48 and Ag(x) antigen), is a large glycoprotein that serves anindispensable role in the assembly and secretion of lipids and in thetransport and receptor-mediated uptake and delivery of distinct classesof lipoproteins. ApoB performs a variety of activities, from theabsorption and processing of dietary lipids to the regulation ofcirculating lipoprotein levels (Davidson and Shelness, Annu. Rev. Nutr.,2000, 20, 169-193). This latter property underlies its relevance interms of atherosclerosis susceptibility, which is highly correlated withthe ambient concentration of ApoB-containing lipoproteins (Davidson andShelness, Annu. Rev. Nutr., 2000, 20, 169-193). ApoB-100 is the majorprotein component of LDL-C and contains the domain required forinteraction of this lipoprotein species with the LDL receptor. Elevatedlevels of LDL-C are a risk factor for cardiovascular disease, includingatherosclerosis.

Definitions

“ApoB” is the gene product or protein of which expression is to bemodulated by administration of a short antisense compound.

“ApoB nucleic acid” means any nucleic acid encoding ApoB. For example,in certain embodiments, a ApoB nucleic acid includes, withoutlimitation, a DNA sequence encoding ApoB, an RNA sequence transcribedfrom DNA encoding ApoB, and an mRNA sequence encoding ApoB.

“ApoB mRNA” means an mRNA encoding ApoB.

ApoB Therapeutic Indications

In certain embodiments, the invention provides methods of modulating theexpression of ApoB in an individual comprising administering a shortantisense compound targeted to an ApoB nucleic acid. In certainembodiments, the invention provides methods of treating an individualcomprising administering one or more pharmaceutical compositionscomprising a short antisense compound targeted to an ApoB nucleic acid.In certain embodiments, the individual has hypercholesterolemia,non-familial hypercholesterolemia, familial hypercholesterolemia,heterozygous familial hypercholesterolemia, homozygous familialhypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk ofdeveloping atherosclerosis, coronary heart disease, a history ofcoronary heart disease, early onset coronary heart disease, one or morerisk factors for coronary heart disease, type II diabetes, type IIdiabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia,hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholicsteatohepatitis, or non-alcoholic fatty liver disease.

Guidelines for lipid-lowering therapy were established in 2001 by AdultTreatment Panel III (ATP III) of the National Cholesterol EducationProgram (NCEP), and updated in 2004 (Grundy et al., Circulation, 2004,110, 227-239). The guidelines include obtaining a complete lipoproteinprofile, typically after a 9 to 12 hour fast, for determination ofLDL-C, total cholesterol, and HDL-C levels. According to the mostrecently established guidelines, LDL-C levels of 130-159 mg/dL, 160-189mg/dL, and greater than or equal to 190 mg/dL are considered borderlinehigh, high, and very high, respectively. Total cholesterol levels of200-239 and greater than or equal to 240 mg/dL are considered borderlinehigh and high, respectively. HDL-C levels of less than 40 mg/dL areconsidered low.

In certain embodiments, the individual has been identified as in need oflipid-lowering therapy. In certain such embodiments, the individual hasbeen identified as in need of lipid-lowering therapy according to theguidelines established in 2001 by Adult Treatment Panel III (ATP III) ofthe National Cholesterol Education Program (NCEP), and updated in 2004(Grundy et al., Circulation, 2004, 110, 227-239). In certain suchembodiments, the individual in need of lipid-lowering therapy has LDL-Cabove 190 mg/dL. In certain such embodiments, the individual in need oflipid-lowering therapy has LDL-C above 160 mg/dL. In certain suchembodiments, the individual in need of lipid-lowering therapy has LDL-Cabove 130 mg/dL. In certain such embodiments the individual in need oflipid-lowering therapy has LDL-C above 100 mg/dL. In certain suchembodiments the individual in need of lipid-lowering therapy shouldmaintain LDL-C below 160 mg/dL. In certain such embodiments theindividual in need of lipid-lowering therapy should maintain LDL-C below130 mg/dL. In certain such embodiments the individual in need oflipid-lowering therapy should maintain LDL-C below 100 mg/dL. In certainsuch embodiments the individual should maintain LDL-C below 70 mg/dL.

In certain embodiments the invention provides methods for reducing ApoBin an individual. In certain embodiments the invention provides methodsfor reducing ApoB-containing lipoprotein in an individual. In certainembodiments the invention provides methods for reducing LDL-C in anindividual. In certain embodiments the invention provides methods forreducing VLDL-C in an individual. In certain embodiments the inventionprovides methods for reducing IDL-C in an individual. In certainembodiments the invention provides methods for reducing non-HDL-C in anindividual. In certain embodiments the invention provides methods forreducing Lp(a) in an individual. In certain embodiments the inventionprovides methods for reducing serum triglyceride in an individual. Incertain embodiments the invention provides methods for reducing livertriglyceride in an individual. In certain embodiments the inventionprovides methods for reducing Ox-LDL-C in an individual. In certainembodiments the invention provides methods for reducing small LDLparticles in an individual. In certain embodiments the inventionprovides methods for reducing small VLDL particles in an individual. Incertain embodiments the invention provides methods for reducingphospholipids in an individual. In certain embodiments the inventionprovides methods for reducing oxidized phospholipids in an individual.

In certain embodiments the invention provides methods for reducingOx-LDL-C concentration in a subject. In certain such embodiments, thereduction in ApoB, LDL-C, VLDL-C, IDL-C, total cholesterol, non-HDL-C,Lp(a), triglycerides, or Ox-LDL-C is, independently, selected from atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, and at least 100%. In certainsuch embodiments, the reduction in ApoB, LDL-C, VLDL-C, IDL-C, totalcholesterol, non-HDL-C, Lp(a), triglycerides, or Ox-LDL-C is,independently, selected from at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, and at least 70%. In certain suchembodiments, the reduction in ApoB, LDL-C, VLDL-C, IDL-C, totalcholesterol, non-HDL-C, Lp(a), triglycerides, or Ox-LDL-C is,independently, selected from at least 40%, at least 50%, at least 60%,and at least 70%.

In certain embodiments, the invention provides method for raising HDL-Cconcentration in a subject.

In certain embodiments, the methods provided by the present invention donot lower HDL-C. In certain embodiments, the methods provided by thepresent invention do not result in accumulation of lipids in the liver.In certain embodiments, the methods provided by the present invention donot cause hepatic steatosis.

In certain embodiments, the invention provides methods for lowering ApoBconcentration in a subject while reducing side effects associated withtreatment. In certain such embodiments, a side effect is liver toxicity.In certain such embodiments, a side effect is abnormal liver function.In certain such embodiments, a side effect is elevated alanineaminotransferase (ALT). In certain such embodiments, a side effect iselevated aspartate aminotransferase (AST).

In certain embodiments, the invention provides methods for lowering ApoBconcentration in a subject who is not reaching target LDL-C levels as aresult of lipid-lowering therapy. In certain such embodiments, a shortantisense compound targeted to an ApoB nucleic acid is the onlylipid-lowering agent administered to the subject. In certain suchembodiments, the subject has not complied with recommendedlipid-lowering therapy. In certain such embodiments, a pharmaceuticalcomposition of the invention is co-administered with an additionaldifferent lipid-lowering therapy. In certain such embodiments, anadditional lipid-lowering therapy is LDL-apheresis. In certain suchembodiments, an additional lipid-lowering therapy is a statin. Incertain such embodiments, an additional lipid-lowering therapy isezetimibe.

In certain embodiments, the invention provides methods for lowering ApoBconcentration in a statin-intolerant subject. In certain suchembodiments, the subject has creatine kinase concentration increases asa result of statin administration. In certain such embodiments, thesubject has liver function abnormalities as a result of statinadministration. In certain such embodiments the subject has muscle achesas a result of statin administration. In certain such embodiments thesubject has central nervous system side effects as a result of statinadministration. In certain embodiments, the subject has not compliedwith recommended statin administration.

In certain embodiments, the invention provides methods for loweringliver triglycerides in a subject. In certain such embodiments, thesubject has elevated liver triglycerides. In certain such embodiments,the subject has steatohepatitis. In certain such embodiments, thesubject has steatosis. In certain such embodiments, liver triglyceridelevels are measured by magnetic resonance imaging.

In certain embodiments, the invention provides methods for reducingcoronary heart disease risk in a subject. In certain embodiments theinvention provides methods for slowing the progression ofatherosclerosis in a subject. In certain such embodiments the inventionprovides methods for stopping the progression of atherosclerosis in asubject. In certain such embodiments the invention provides methods forreducing the size and/or prevalence of atherosclerotic plaques in asubject. In certain embodiments the methods provided reduce a subject'srisk of developing atherosclerosis.

In certain embodiments the methods provided improve the cardiovascularoutcome in a subject. In certain such embodiments improvedcardiovascular outcome is the reduction of the risk of developingcoronary heart disease. In certain such embodiments, improvedcardiovascular outcome is a reduction in the occurrence of one or moremajor cardiovascular events, which include, but are not limited to,death, myocardial infarction, reinfarction, stroke, cardiogenic shock,pulmonary edema, cardiac arrest, and atrial dysrhythmia. In certain suchembodiments, the improved cardiovascular outcome is evidenced byimproved carotid intimal media thickness. In certain such embodiments,improved carotid intimal media thickness is a decrease in thickness. Incertain such embodiments, improved carotid intimal media thickness is aprevention an increase of intimal media thickness.

In certain embodiments a pharmaceutical composition comprising a shortantisense compound targeted to an ApoB nucleic acid is for use intherapy. In certain embodiments, the therapy is the reduction of LDL-C,ApoB, VLDL-C, IDL-C, non-HDL-C, Lp(a), serum triglyceride, livertriglyceride, Ox-LDL-C, small LDL particles, small VLDL, phospholipids,or oxidized phospholipids in an individual. In certain embodiments, thetherapy is the treatment of hypercholesterolemia, non-familialhypercholesterolemia, familial hypercholesterolemia, heterozygousfamilial hypercholesterolemia, homozygous familial hypercholesterolemia,mixed dyslipidemia, atherosclerosis, a risk of developingatherosclerosis, coronary heart disease, a history of coronary heartdisease, early onset coronary heart disease, one or more risk factorsfor coronary heart disease, type II diabetes, type II diabetes withdyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia,hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis, ornon-alcoholic fatty liver disease. In additional embodiments, thetherapy is the reduction of CHD risk. In certain the therapy isprevention of atherosclerosis. In certain embodiments, the therapy isthe prevention of coronary heart disease.

In certain embodiments a pharmaceutical composition comprising a shortantisense compound targeted to an ApoB nucleic acid is used for thepreparation of a medicament for reducing LDL-C, ApoB, VLDL-C, IDL-C,non-HDL-C, Lp(a), serum triglyceride, liver triglyceride, Ox-LDL-C,small LDL particles, small VLDL, phospholipids, or oxidizedphospholipids in an individual. In certain embodiments pharmaceuticalcomposition comprising a short antisense compound targeted to an ApoBnucleic acid is used for the preparation of a medicament for reducingcoronary heart disease risk. In certain embodiments a short antisensecompound targeted to an ApoB nucleic acid is used for the preparation ofa medicament for the treatment of hypercholesterolemia, non-familialhypercholesterolemia, familial hypercholesterolemia, heterozygousfamilial hypercholesterolemia, homozygous familial hypercholesterolemia,mixed dyslipidemia, atherosclerosis, a risk of developingatherosclerosis, coronary heart disease, a history of coronary heartdisease, early onset coronary heart disease, one or more risk factorsfor coronary heart disease, type II diabetes, type II diabetes withdyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia,hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis, ornon-alcoholic fatty liver disease.

ApoB Combination Therapies

In certain embodiments, one or more pharmaceutical compositionscomprising a short antisense compound targeted to an ApoB nucleic acidare co-administered with one or more other pharmaceutical agents. Incertain embodiments, such one or more other pharmaceutical agents aredesigned to treat the same disease or condition as the one or morepharmaceutical compositions of the present invention. In certain suchembodiments, the one or more pharmaceutical agents are lipid-loweringagents. In certain embodiments, such one or more other pharmaceuticalagents are designed to treat a different disease or condition as the oneor more pharmaceutical compositions of the present invention. In certainembodiments, such one or more other pharmaceutical agents are designedto treat an undesired effect of one or more pharmaceutical compositionsof the present invention. In certain embodiments, one or morepharmaceutical compositions of the present invention are co-administeredwith another pharmaceutical agent to treat an undesired effect of thatother pharmaceutical agent. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are administered at the same time. Incertain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areadministered at different times. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are prepared together in a singleformulation. In certain embodiments, one or more pharmaceuticalcompositions of the present invention and one or more otherpharmaceutical agents are prepared separately.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition comprising a shortantisense compound targeted to an ApoB nucleic acid includelipid-lowering agents. In certain such embodiments, pharmaceuticalagents that may be co-administered with a pharmaceutical composition ofthe present invention include, but are not limited to atorvastatin,simvastatin, rosuvastatin, and ezetimibe. In certain such embodiments,the lipid-lowering agent is administered prior to administration of apharmaceutical composition of the present invention. In certain suchembodiments, the lipid-lowering agent is administered followingadministration of a pharmaceutical composition of the present invention.In certain such embodiments the lipid-lowering agent is administered atthe same time as a pharmaceutical composition of the present invention.In certain such embodiments the dose of a co-administered lipid-loweringagent is the same as the dose that would be administered if thelipid-lowering agent was administered alone. In certain such embodimentsthe dose of a co-administered lipid-lowering agent is lower than thedose that would be administered if the lipid-lowering agent wasadministered alone. In certain such embodiments the dose of aco-administered lipid-lowering agent is greater than the dose that wouldbe administered if the lipid-lowering agent was administered alone.

In certain embodiments, a co-administered lipid-lowering agent is aHMG-CoA reductase inhibitor. In certain such embodiments the HMG-CoAreductase inhibitor is a statin. In certain such embodiments the statinis selected from atorvastatin, simvastatin, pravastatin, fluvastatin,and rosuvastatin.

In certain embodiments, a co-administered lipid-lowering agent is acholesterol absorption inhibitor. In certain such embodiments,cholesterol absorption inhibitor is ezetimibe.

In certain embodiments, a co-administered lipid-lowering agent is aco-formulated HMG-CoA reductase inhibitor and cholesterol absorptioninhibitor. In certain such embodiments the co-formulated lipid-loweringagent is ezetimibe/simvastatin.

In certain embodiments, a co-administered lipid-lowering agent is amicrosomal triglyceride transfer protein inhibitor (MTP inhibitor).

In certain embodiments, a co-administered pharmaceutical agent is a bileacid sequestrant. In certain such embodiments, the bile acid sequestrantis selected from cholestyramine, colestipol, and colesevelam.

In certain embodiments, a co-administered pharmaceutical agent is anicotinic acid. In certain such embodiments, the nicotinic acid isselected from immediate release nicotinic acid, extended releasenicotinic acid, and sustained release nicotinic acid.

In certain embodiments, a co-administered pharmaceutical agent is afibric acid. In certain such embodiments, a fibric acid is selected fromgemifibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.

Further examples of pharmaceutical agents that may be co-administeredwith a pharmaceutical composition comprising a short antisense compoundtargeted to an ApoB nucleic acid include, but are not limited to,corticosteroids, including but not limited to prednisone;immunoglobulins, including, but not limited to intravenousimmunoglobulin (IVIg); analgesics (e.g., acetaminophen);anti-inflammatory agents, including, but not limited to non-steroidalanti-inflammatory drugs (e.g., ibuprofen, COX-1 inhibitors, and COX-2,inhibitors); salicylates; antibiotics; antivirals; antifungal agents;antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins,sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics;hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin,progestin, somatostan, and thyroid hormones); immunomodulators; musclerelaxants; antihistamines; osteoporosis agents (e.g., biphosphonates,calcitonin, and estrogens); prostaglandins, antineoplastic agents;psychotherapeutic agents; sedatives; poison oak or poison sumacproducts; antibodies; and vaccines.

In certain embodiments, a pharmaceutical composition comprising a shortantisense compound targeted to an ApoB nucleic acid may be administeredin conjunction with a lipid-lowering therapy. In certain suchembodiments, a lipid-lowering therapy is therapeutic lifestyle change.In certain such embodiments, a lipid-lowering therapy is LDL apheresis.

In one embodiment, the antisense compounds provided herein can be usedto lower the level of apolipoprotein B-containing lipoproteins in ahuman subject. As used herein, “apolipoprotein B-containing lipoprotein”refers to any lipoprotein that has apolipoprotein B as its proteincomponent, and is understood to include LDL, VLDL, IDL, andlipoprotein(a). LDL, VLDL, IDL and lipoprotein(a) each contain onemolecule of apolipoprotein B, thus a serum apolipoprotein B measurementreflects the total number of these lipoproteins. As is known in the art,each of the aforementioned lipoproteins is atherogenic. Thus, loweringone or more apolipoprotein B-containing lipoproteins in serum mayprovide a therapeutic benefit to a human subject. Small LDL particlesare considered to be particularly atherogenic relative to large LDLparticles, thus lowering small LDL particles can provide a therapeuticbenefit to a human subject. Additional lipid parameters can also bedetermined in a subject. Reduction of total cholesterol:HDL ratio orLDL:HDL ratio is a clinically desirable improvement in cholesterolratio. Similarly, it is clinically desirable to reduce serumtriglycerides in humans who exhibit elevated lipid levels.

Other indications of cardiovascular disease that can be measured in asubject include serum LDL particle size; serum LDL cholesteryl esterconcentration; serum LDL cholesteryl ester composition; the extent ofpolyunsaturation of serum LDL cholesteryl esters; and serum HDLcholesterol levels. As used herein, “serum LDL particle size” refers tothe classification of serum LDL particle size, which may be very small,small, medium, or large, and is typically expressed in g/μmol. In thecontext of the present invention, “serum LDL cholesteryl esterconcentration” means the amount of cholesteryl ester present in LDLparticles, and is typically measured as mg/dL. In the context of thepresent invention, “serum LDL cholesteryl ester composition” is ameasurement of the percentage of saturated, monounsaturated andpolyunsaturated cholesteryl ester fatty acids present in serum LDLparticles. “Polyunsaturation of serum LDL cholesteryl esters” means thepercentage of polyunsaturated cholesteryl ester fatty acids in serum LDLparticles.

Methods of obtaining serum or plasma samples for analysis and methods ofpreparation of the serum samples to allow for analysis are well known tothose skilled in the art. With regard to measurements of lipoproteins,cholesterol, triglyceride and cholesteryl esters, the terms “serum” and“plasma” are herein used interchangeably.

In another embodiment, the antisense compounds provided herein can beused to treat metabolic disorders. A variety of biomarkers can be usedfor evaluating metabolic disease. For example, blood glucose levels canbe determined by a physician or even by the patient using a commonlyavailable test kit or glucometer (for example, the Ascensia ELITE™ kit,Ascensia (Bayer), Tarrytown N.Y., or Accucheck, Roche Diagnostics).Glycated hemoglobin (HbA_(1c)) can also be measured. HbA_(1c) is astable minor hemoglobin variant formed in vivo via posttranslationalmodification by glucose, and it contains predominantly glycatedN₂-terminal β-chains. There is a strong correlation between levels ofHbA_(1c) and the average blood glucose levels over the previous 3months. Thus HbA_(1c) is often viewed as the “gold standard” formeasuring sustained blood glucose control (Bunn, H. F. et al., 1978,Science. 200, 21-7). HbA_(1c) can be measured by ion-exchange HPLC orimmunoassay; home blood collection and mailing kits for HbA_(1c)measurement are now widely available. Serum fructosamine is anothermeasure of stable glucose control and can be measured by a colorimetricmethod (Cobas Integra, Roche Diagnostics).

Certain Short Antisense Compounds Targeted to an ApoB Nucleic Acid

In certain embodiments, short antisense compounds are targeted to anApoB nucleic acid having the sequence of GENBANK® Accession No.NM-000384.1, incorporated herein as SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to SEQ ID NO: 1 is atleast 90% complementary to SEQ ID NO: 1. In certain such embodiments, ashort antisense compound targeted to SEQ ID NO: 1 is at least 95%complementary to SEQ ID NO: 1. In certain such embodiments, a shortantisense compound targeted to SEQ ID NO: 1 is 100% complementary to SEQID NO: 1. In certain embodiments, a short antisense compound targeted toSEQ ID NO: 1 comprises a nucleotide sequence selected from thenucleotide sequences set forth in Table 2 and Table 3.

The nucleotide sequence set forth in each SEQ ID NO in Tables 2 and 3 isindependent of any modification to a sugar moiety, a monomeric linkage,or a nucleobase. As such, short antisense compounds defined by a SEQ IDNO may comprise, independently, one or more modifications to a sugarmoiety, an internucleoside linkage, or a nucleobase. Antisense compoundsdescribed by Isis Number (Isis NO.) indicate a combination of nucleobasesequence and one or more modifications to a sugar moiety, aninternucleoside linkage, or a nucleobase.

Tables 2 and 3 illustrate examples of short antisense compounds targetedto SEQ ID NO: 1. Table 2 illustrates short antisense compounds that are100% complementary to SEQ ID NO: 1. Table 3 illustrates short antisensecompounds that have one or two mismatches with respect to SEQ ID NO: 1.The column labeled ‘gapmer motif’ indicates the wing-gap-wing motif ofeach short antisense compounds. The gap segment comprises2′-deoxynucleotides and each nucleotide of each wing segment comprises a2′-modified sugar. The particular 2′-modified sugar is also indicated inthe ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flankedby wing segments of two nucleotides, where the nucleotides of the wingsegments are 2′-MOE nucleotides. Internucleoside linkages arephosphorothioate. The short antisense compounds comprise5-methylcytidine in place of unmodified cytosine, unless “unmodifiedcytosine” is listed in the gapmer motif column, in which case theindicated cytosines are unmodified cytosines. For example, “5-mC in gaponly” indicates that the gap segment has 5-methylcytosines, while thewing segments have unmodified cytosines.

TABLE 2 Short Antisense Compounds targeted to SEQ ID NO: 1 5′ 3′ ISISTarget Target SEQ No Site Site Sequence (5′-3′) Gapmer Motif ID NO372816 263 278 CCGGAGGTGCTTGAAT 3-10-3 MOE 16 372894 264 277CGGAGGTGCTTGAA 2-10-2 MOE 17 372817 428 443 GAAGCCATACACCTCT 3-10-3 MOE18 372895 429 442 AAGCCATACACCTC 2-10-2 MOE 19 372818 431 446GTTGAAGCCATACACC 3-10-3 MOE 20 372896 432 445 TTGAAGCCATACAC 2-10-2 MOE21 372819 438 453 CCTCAGGGTTGAAGCC 3-10-3 MOE 22 372897 439 452CTCAGGGTTGAAGC 2-10-2 MOE 23 372820 443 458 TTTGCCCTCAGGGTTG 3-10-3 MOE24 372898 444 457 TTGCCCTCAGGGTT 2-10-2 MOE 25 372821 468 483AGTTCTTGGTTTTCTT 3-10-3 MOE 26 372899 469 482 GTTCTTGGTTTTCT 2-10-2 MOE27 372822 587 602 CCTCTTGATGTTCAGG 3-10-3 MOE 28 372900 588 601CTCTTGATGTTCAG 2-10-2 MOE 29 372823 592 607 ATGCCCCTCTTGATGT 3-10-3 MOE30 372901 593 606 TGCCCCTCTTGATG 2-10-2 MOE 31 346583 715 728TGCCACATTGCCCT 3-8-3 MOE 32 346584 716 729 TTGCCACATTGCCC 3-8-3 MOE 33346585 717 730 GTTGCCACATTGCC 3-8-3 MOE 34 346586 718 731 TGTTGCCACATTGC3-8-3 MOE 35 346587 719 732 CTGTTGCCACATTG 3-8-3 MOE 36 346588 720 733TCTGTTGCCACATT 3-8-3 MOE 37 346589 721 734 TTCTGTTGCCACAT 3-8-3 MOE 38346590 722 735 TTTCTGTTGCCACA 3-8-3 MOE 39 346591 723 736 ATTTCTGTTGCCAC3-8-3 MOE 40 372824 929 944 GTAGGAGAAAGGCAGG 3-10-3 MOE 41 372902 930943 TAGGAGAAAGGCAG 2-10-2 MOE 42 372825 1256 1271 GGCTTGTAAAGTGATG3-10-3 MOE 43 372903 1257 1270 GCTTGTAAAGTGAT 2-10-2 MOE 44 372826 13041319 CCACTGGAGGATGTGA 3-10-3 MOE 45 372904 1305 1318 CACTGGAGGATGTG2-10-2 MOE 46 372829 2135 2150 TTTCAGCATGCTTTCT 3-10-3 MOE 47 3729072136 2149 TTCAGCATGCTTTC 2-10-2 MOE 48 372832 2774 2789 CATATTTGTCACAAAC3-10-3 MOE 49 372910 2775 2788 ATATTTGTCACAAA 2-10-2 MOE 50 372833 27792794 ATGCCCATATTTGTCA 3-10-3 MOE 51 372911 2780 2793 TGCCCATATTTGTC2-10-2 MOE 52 372835 2961 2976 TTTTGGTGGTAGAGAC 3-10-3 MOE 53 3729132962 2975 TTTGGTGGTAGAGA 2-10-2 MOE 54 346592 3248 3261 TCTGCTTCGCACCT3-8-3 MOE 55 346593 3249 3262 GTCTGCTTCGCACC 3-8-3 MOE 56 346594 32503263 AGTCTGCTTCGCAC 3-8-3 MOE 57 346595 3251 3264 CAGTCTGCTTCGCA 3-8-3MOE 58 346596 3252 3265 TCAGTCTGCTTCGC 3-8-3 MOE 59 346597 3253 3266CTCAGTCTGCTTCG 3-8-3 MOE 60 346598 3254 3267 CCTCAGTCTGCTTC 3-8-3 MOE 61346599 3255 3268 GCCTCAGTCTGCTT 3-8-3 MOE 62 346600 3256 3269AGCCTCAGTCTGCT 3-8-3 MOE 63 372836 3350 3365 AACTCTGAGGATTGTT 3-10-3 MOE64 372914 3351 3364 ACTCTGAGGATTGT 2-10-2 MOE 65 372837 3355 3370TCATTAACTCTGAGGA 3-10-3 MOE 66 372915 3356 3369 CATTAACTCTGAGG 2-10-2MOE 67 372838 3360 3375 ATTCATCATTAACTCT 3-10-3 MOE 68 372916 3361 3374TTCATCATTAACTC 2-10-2 MOE 69 372839 3409 3424 TTGTTCTGAATGTCCA 3-10-3MOE 70 387461 3409 3424 TTGTTCTGAATGTCCA 3-10-3 Methyleneoxy 70 BNAUnmodified cytosines in gap 380147 3409 3424 TTGTTCTGAATGTCCA 3-10-3Methyleneoxy 70 BNA 372917 3410 3423 TGTTCTGAATGTCC 2-10-2 MOE 73 3728403573 3588 CAGATGAGTCCATTTG 3-10-3 MOE 74 372918 3574 3587 AGATGAGTCCATTT2-10-2 MOE 75 372841 3701 3716 ATCCACAGGGAAATTG 3-10-3 MOE 76 3729193702 3715 TCCACAGGGAAATT 2-10-2 MOE 77 372843 4219 4234 CAGTTGTACAAGTTGC3-10-3 MOE 78 372921 4220 4233 AGTTGTACAAGTTG 2-10-2 MOE 79 372844 43014316 CACAGAGTCAGCCTTC 3-10-3 MOE 80 372922 4302 4315 ACAGAGTCAGCCTT2-10-2 MOE 81 372845 4308 4323 GGTCAACCACAGAGTC 3-10-3 MOE 82 3729234309 4322 GTCAACCACAGAGT 2-10-2 MOE 83 346601 5588 5601 CAGCCACATGCAGC3-8-3 MOE 84 346602 5589 5602 CCAGCCACATGCAG 3-8-3 MOE 85 346603 55905603 ACCAGCCACATGCA 3-8-3 MOE 86 346604 5591 5604 TACCAGCCACATGC 3-8-3MOE 87 346605 5592 5605 TTACCAGCCACATG 3-8-3 MOE 88 346606 5593 5606GTTACCAGCCACAT 3-8-3 MOE 89 346607 5594 5607 GGTTACCAGCCACA 3-8-3 MOE 90346608 5595 5608 AGGTTACCAGCCAC 3-8-3 MOE 91 346609 5596 5609TAGGTTACCAGCCA 3-8-3 MOE 92 372851 5924 5939 AGGTTCTGCTTTCAAC 3-10-3 MOE93 372929 5925 5938 GGTTCTGCTTTCAA 2-10-2 MOE 94 372854 6664 6679TACTGATCAAATTGTA 3-10-3 MOE 95 372932 6665 6678 ACTGATCAAATTGT 2-10-2MOE 96 372855 6908 6923 TTTTTCTTGTATCTGG 3-10-3 MOE 97 372933 6909 6922TTTTCTTGTATCTG 2-10-2 MOE 98 372856 7190 7205 ATCCATTAAAACCTGG 3-10-3MOE 99 372934 7191 7204 TCCATTAAAACCTG 2-10-2 MOE 100 372858 7817 7832ATATTGCTCTGCAAAG 3-10-3 MOE 101 372936 7818 7831 TATTGCTCTGCAAA 2-10-2MOE 102 346610 7818 7831 TATTGCTCTGCAAA 3-8-3 MOE 102 346611 7819 7832ATATTGCTCTGCAA 3-8-3 MOE 104 346612 7820 7833 AATATTGCTCTGCA 3-8-3 MOE105 346613 7821 7834 GAATATTGCTCTGC 3-8-3 MOE 106 346614 7822 7835AGAATATTGCTCTG 3-8-3 MOE 107 346615 7823 7836 TAGAATATTGCTCT 3-8-3 MOE108 346616 7824 7837 ATAGAATATTGCTC 3-8-3 MOE 109 346617 7825 7838GATAGAATATTGCT 3-8-3 MOE 110 346618 7826 7839 GGATAGAATATTGC 3-8-3 MOE111 372859 7995 8010 ATGGAATCCTCAAATC 3-10-3 MOE 112 372937 7996 8009TGGAATCCTCAAAT 2-10-2 MOE 113 372861 8336 8351 GAATTCTGGTATGTGA 3-10-3MOE 114 372939 8337 8350 AATTCTGGTATGTG 2-10-2 MOE 115 372862 8341 8356AGCTGGAATTCTGGTA 3-10-3 MOE 116 372940 8342 8355 GCTGGAATTCTGGT 2-10-2MOE 117 372863 8539 8554 TGAAAATCAAAATTGA 3-10-3 MOE 118 372941 85408553 GAAAATCAAAATTG 2-10-2 MOE 119 372871 9344 9359 AAACAGTGCATAGTTA3-10-3 MOE 120 372949 9345 9358 AACAGTGCATAGTT 2-10-2 MOE 121 3728729515 9530 TTCAGGAATTGTTAAA 3-10-3 MOE 122 372950 9516 9529TCAGGAATTGTTAA 2-10-2 MOE 123 372875 9794 9809 TTTTGTTTCATTATAG 3-10-3MOE 124 372953 9795 9808 TTTGTTTCATTATA 2-10-2 MOE 125 372877 1015710172 GATGACACTTGATTTA 3-10-3 MOE 126 372955 10158 10171 ATGACACTTGATTT2-10-2 MOE 127 372878 10161 10176 GTGTGATGACACTTGA 3-10-3 MOE 128 37295610162 10175 TGTGATGACACTTG 2-10-2 MOE 129 372879 10167 10182TATTCAGTGTGATGAC 3-10-3 MOE 130 372957 10168 10181 ATTCAGTGTGATGA 2-10-2MOE 131 372880 10172 10187 ATTGGTATTCAGTGTG 3-10-3 MOE 132 372958 1017310186 TTGGTATTCAGTGT 2-10-2 MOE 133 346619 10838 10851 CCTCTAGCTGTAAG3-8-3 MOE 134 346620 10839 10852 CCCTCTAGCTGTAA 3-8-3 MOE 135 34662110840 10853 GCCCTCTAGCTGTA 3-8-3 MOE 136 346622 10841 10854GGCCCTCTAGCTGT 3-8-3 MOE 137 346623 10842 10855 AGGCCCTCTAGCTG 3-8-3 MOE138 346624 10843 10856 GAGGCCCTCTAGCT 3-8-3 MOE 139 346625 10844 10857AGAGGCCCTCTAGC 3-8-3 MOE 140 346626 10845 10858 AAGAGGCCCTCTAG 3-8-3 MOE141 346627 10846 10859 AAAGAGGCCCTCTA 3-8-3 MOE 142 372890 13689 13704GAATGGACAGGTCAAT 3-10-3 MOE 143 372968 13690 13703 AATGGACAGGTCAA 2-10-2MOE 144 372891 13694 13709 GTTTTGAATGGACAGG 3-10-3 MOE 145 372969 1369513708 TTTTGAATGGACAG 2-10-2 MOE 146 372892 13699 13714 TGGTAGTTTTGAATGG3-10-3 MOE 147 372970 13700 13713 GGTAGTTTTGAATG 2-10-2 MOE 148 34662813907 13920 TCACTGTATGGTTT 3-8-3 MOE 149 346629 13908 13921CTCACTGTATGGTT 3-8-3 MOE 150 346630 13909 13922 GCTCACTGTATGGT 3-8-3 MOE151 346631 13910 13923 GGCTCACTGTATGG 3-8-3 MOE 152 346632 13911 13924TGGCTCACTGTATG 3-8-3 MOE 153 346633 13912 13925 CTGGCTCACTGTAT 3-8-3 MOE154 346634 13913 13926 GCTGGCTCACTGTA 3-8-3 MOE 155 346635 13914 13927GGCTGGCTCACTGT 3-8-3 MOE 156 346636 13915 13928 AGGCTGGCTCACTG 3-8-3 MOE157 346637 13963 13976 CAGGTCCAGTTCAT 3-8-3 MOE 158 346638 13964 13977GCAGGTCCAGTTCA 3-8-3 MOE 159 346639 13965 13978 TGCAGGTCCAGTTC 3-8-3 MOE160 346640 13966 13979 GTGCAGGTCCAGTT 3-8-3 MOE 161 346641 13967 13980GGTGCAGGTCCAGT 3-8-3 MOE 162 346642 13968 13981 TGGTGCAGGTCCAG 3-8-3 MOE163 346643 13969 13982 TTGGTGCAGGTCCA 3-8-3 MOE 164 346644 13970 13983TTTGGTGCAGGTCC 3-8-3 MOE 165 346645 13971 13984 CTTTGGTGCAGGTC 3-8-3 MOE166 346646 14051 14064 TAACTCAGATCCTG 3-8-3 MOE 167 346647 14052 14065ATAACTCAGATCCT 3-8-3 MOE 168 346648 14053 14066 AATAACTCAGATCC 3-8-3 MOE169 346649 14054 14067 AAATAACTCAGATC 3-8-3 MOE 170 346650 14055 14068AAAATAACTCAGAT 3-8-3 MOE 171 346651 14056 14069 CAAAATAACTCAGA 3-8-3 MOE172 346652 14057 14070 GCAAAATAACTCAG 3-8-3 MOE 173 346653 14058 14071AGCAAAATAACTCA 3-8-3 MOE 174 346654 14059 14072 TAGCAAAATAACTC 3-8-3 MOE175

TABLE 3 Short antisense compounds targeted to SEQ ID NO: 1 and having 1or 2 mismatches 5′ 3′ SEQ Isis Target Target ID NO. Site Site Sequence(5′-3′) Gapmer Motif NO 372894 771 784 CGGAGGTGCTTGAA 2-10-2 MOE 17372905 1111 1124 CAGGGCCTGGAGAG 2-10-2 MOE 176 346628 1493 1506TCACTGTATGGTTT 3-8-3 MOE 149 372828 2006 2021 TCTGAAGTCCATGATC 3-10-3MOE 177 372906 2007 2020 CTGAAGTCCATGAT 2-10-2 MOE 178 372830 2382 2397TGGGCATGATTCCATT 3-10-3 MOE 179 372908 2383 2396 GGGCATGATTCCAT 2-10-2MOE 180 346616 3162 3175 ATAGAATATTGCTC 3-8-3 MOE 109 346617 3163 3176GATAGAATATTGCT 3-8-3 MOE 110 372929 3513 3526 GGTTCTGCTTTCAA 2-10-2 MOE94 372946 3800 3813 TGGAGCCCACGTGC 2-10-2 MOE 181 372904 4040 4053CACTGGAGGATGTG 2-10-2 MOE 46 372842 4084 4099 TTGAAGTTGAGGGCTG 3-10-3MOE 182 372920 4085 4098 TGAAGTTGAGGGCT 2-10-2 MOE 183 346586 4778 4791TGTTGCCACATTGC 3-8-3 MOE 35 372847 5030 5045 ACCAGTATTAATTTTG 3-10-3 MOE184 372925 5031 5044 CCAGTATTAATTTT 2-10-2 MOE 185 372848 5192 5207GTGTTCTTTGAAGCGG 3-10-3 MOE 186 372926 5193 5206 TGTTCTTTGAAGCG 2-10-2MOE 187 372953 5625 5638 TTTGTTTCATTATA 2-10-2 MOE 125 372935 7585 7598AGTTACTTTGGTGT 2-10-2 MOE 188 372860 8255 8270 TGGTACATGGAAGTCT 3-10-3MOE 189 372938 8256 8269 GGTACATGGAAGTC 2-10-2 MOE 190 391260 8256 8269GGTACATGGAAGTC 2-10-2 MOE 190 392068 8256 8269 GGTACATGGAAGTC 2-10-2 MOE190 387462 8256 8269 GGTACATGGAAGTC 2-10-2 Methyleneoxy 190 BNA 3918728256 8269 GGTACATGGAAGTC 1-1-10-2 2′- 190 (butylacetomido)- palmitamideMethyleneoxy BNA/Methyleneoxy BNA Unmodified cytosines in gap 3801488256 8269 GGTACATGGAAGTC 2-10-2 Methyleneoxy 190 BNA 391871 8256 8269GGTACATGGAAGTC 1-1-10-2 2′- 190 (butylacetomido)- palmitamide/MOE/MOEUnmodified cytosines in gap 391755 8256 8269 GGTACATGGAAGTC 2-10-2 ENA190 mC in wing only 398296 8256 8269 GGTACATGGAAGTC 2-10-2(6′S)-6′-methyl- 190 Methyleneoxy BNA Unmodified Cytosines 372942 84558468 TCCATGCCATATGT 2-10-2 MOE 200 372865 8888 8903 CCCTGAAGAAGTCCAT3-10-3 MOE 201 372943 8889 8902 CCTGAAGAAGTCCA 2-10-2 MOE 202 3728668908 8923 GCCCAGTTCCATGACC 3-10-3 MOE 203 372944 8909 8922CCCAGTTCCATGAC 2-10-2 MOE 204 372867 9058 9073 TTGAGGAAGCCAGATT 3-10-3MOE 205 372945 9059 9072 TGAGGAAGCCAGAT 2-10-2 MOE 206 372870 9261 9276TGGATGCAGTAATCTC 3-10-3 MOE 207 372948 9262 9275 GGATGCAGTAATCT 2-10-2MOE 208 372881 10185 10200 TATAAAGTCCAGCATT 3-10-3 MOE 209 372959 1018610199 ATAAAGTCCAGCAT 2-10-2 MOE 210 372882 10445 10460 AAGTTCCTGCTTGAAG3-10-3 MOE 211 372960 10446 10459 AGTTCCTGCTTGAA 2-10-2 MOE 212 37296411451 11464 AATGGTGAAGTACT 2-10-2 MOE 213 346612 13459 13472AATATTGCTCTGCA 3-8-3 MOE 105 346613 13460 13473 GAATATTGCTCTGC 3-8-3 MOE106

In certain embodiments, a target region is nucleotides 263-278 of SEQ IDNO: 1. In certain such embodiments, short antisense compounds targetedto nucleotides 263-278 of SEQ ID NO: 1 comprise a nucleotide sequenceselected from SEQ ID NO: 16 or 17. In certain such embodiments, a shortantisense compound targeted to nucleotides 263-278 of SEQ ID NO: 1 isselected from Isis NO. 372816 or 372894.

In certain embodiments, a target region is nucleotides 428-483 of SEQ IDNO: 1. In certain such embodiments, a short antisense compound targetedto nucleotides 428-483 of SEQ ID NO: 1 comprises a nucleotide sequenceselected from SEQ ID NO 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27. Incertain such embodiments, a short antisense compound targeted tonucleotides 428-483 of SEQ ID NO: 1 is selected from Isis NO. 372817,372895, 372818, 372896, 372819, 372897, 372820, 372898, 372821, or372899.

In certain embodiments, a target region is nucleotides 428-458 of SEQ IDNO: 1. In certain such embodiments, a short antisense compound targetedto nucleotides 428-458 of SEQ ID NO: 1 comprises a nucleotide sequenceselected from SEQ ID NO 18, 19, 20, 21, 22, 23, 24, or 25. In certainsuch embodiments, a short antisense compound targeted to nucleotides428-458 of SEQ ID NO: 1 is selected from Isis NO. 372817, 372895,372818, 372896, 372819, 372897, 372820, or 372898.

In certain embodiments, a target region is nucleotides 468-483 of SEQ IDNO: 1. In certain such embodiments, a short antisense compound targetedto nucleotides 468-483 of SEQ ID NO: 1 comprises a nucleotide sequenceselected from SEQ ID NO 26 or 27. In certain such embodiments, a shortantisense compound targeted to nucleotides 468-483 of SEQ ID NO: 1 isselected from Isis NO. 372821 or 372899.

In certain embodiments, a target region is nucleotides 587-607 of SEQ IDNO: 1. In certain such embodiments, a short antisense compound targetedto nucleotides 587-607 of SEQ ID NO: 1 comprises a nucleotide sequenceselected from SEQ ID NO 28, 29, 30, or 31. In certain such embodiments,a short antisense compound targeted to nucleotides 587-607 of SEQ ID NO:1 is selected from ISIS NO. 372822, 372900, 372823, or 372901.

In certain embodiments, a target region is nucleotides 715-736 of SEQ IDNO: 1. In certain such embodiments, a short antisense compound targetedto nucleotides 715-736 of SEQ ID NO: 1 comprises a nucleotide sequenceselected from SEQ ID NO 32, 33, 34, 35, 36, 37, 38, 39, or 40. Incertain such embodiments, a short antisense compound targeted tonucleotides 715-736 of SEQ ID NO: 1 is selected from Isis NO. 346583,346584, 346585, 346586, 346587, 346588, 346589, 346590, or 346591.

In certain embodiments, a target region is nucleotides 929-944 of SEQ IDNO: 1. In certain such embodiments, a short antisense compound targetedto nucleotides 929-944 of SEQ ID NO: 1 comprises a nucleotide sequenceselected from SEQ ID NO 41 or 42. In certain such embodiments, a shortantisense compound targeted to nucleotides 929-944 of SEQ ID NO: 1 isselected from Isis NO. 372824 or 372902.

In certain embodiments, a target region is nucleotides 1256-1319 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1256-1319 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 43, 44, 45, or 46. In certain suchembodiments, a short antisense compound targeted to nucleotides1256-1319 of SEQ ID NO: 1 is selected from Isis NO. 372825, 372903,372826, or 372904.

In certain embodiments, a target region is nucleotides 1256-1271 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1256-1271 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 43 or 44. In certain such embodiments,a short antisense compound targeted to nucleotides 1256-1271 of SEQ IDNO: 1 is selected from Isis NO. 372825 or 372903.

In certain embodiments, a target region is nucleotides 1304-1319 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1304-1319 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 45 or 46. In certain such embodiments,a short antisense compound targeted to nucleotides 1304-1319 of SEQ IDNO: 1 is selected from Isis NO. 372826 or 372904.

In certain embodiments, a target region is nucleotides 2135-2150 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 2135-2150 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 47 or 48. In certain such embodiments,a short antisense compound targeted to nucleotides 2135-2150 of SEQ IDNO: 1 is selected from ISIS NO. 372829 or 372907.

In certain embodiments, a target region is nucleotides 2774-2794 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 2774-2794 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 49, 50, 51, or 52. In certain suchembodiments, a short antisense compound targeted to nucleotides2774-2794 of SEQ ID NO: 1 is selected from ISIS NO. 372832, 372910,372833, or 372911.

In certain embodiments, a target region is nucleotides 2961-2976 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 2961-2976 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 53 or 54. In certain such embodiments,a short antisense compound targeted to nucleotides 2961-2976 of SEQ IDNO: 1 is selected from ISIS NO. 372835 or 372913.

In certain embodiments, a target region is nucleotides 3248-3269 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 3248-3269 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 55, 56, 57, 58, 59, 60, 61, 62, or 63.In certain such embodiments, a short antisense compound targeted tonucleotides 3248-3269 of SEQ ID NO: 1 is selected from ISIS NO. 346592,346593, 346594, 346595, 346596, 346597, 346598, 346599, or 346600.

In certain embodiments, a target region is nucleotides 3350-3375 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 3350-3375 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 64, 65, 66, 67, 68, or 69. In certainsuch embodiments, a short antisense compound targeted to nucleotides3350-3375 of SEQ ID NO: 1 is selected from ISIS NO. 372836, 372914,372837, 372915, 372838, or 372916.

In certain embodiments, a target region is nucleotides 3409-3424 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 3409-3424 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 70 or 73. In certain such embodiments,a short antisense compound targeted to nucleotides 3409-3424 of SEQ IDNO: 1 is selected from ISIS NO. 372839, 387461, 380147, or 372917.

In certain embodiments, a target region is nucleotides 3573-3588 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 3573-3588 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 74 or 75. In certain such embodiments,a short antisense compound targeted to nucleotides 3573-3588 of SEQ IDNO: 1 is selected from ISIS NO. 372840 or 372918.

In certain embodiments, a target region is nucleotides 3701-3716 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 3701-3716 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 76 or 77. In certain such embodiments,a short antisense compound targeted to nucleotides 3701-3716 of SEQ IDNO: 1 is selected from ISIS NO. 372841 or 372919.

In certain embodiments, a target region is nucleotides 4219-4234 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 4219-4234 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 78 or 79. In certain such embodiments,a short antisense compound targeted to nucleotides 4219-4234 of SEQ IDNO: 1 is selected from ISIS NO. 372843 or 372921.

In certain embodiments, a target region is nucleotides 4301-4323 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 4301-4323 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 80, 81, 82, or 83. In certainembodiments, a short antisense compound targeted to nucleotides4301-4323 of SEQ ID NO: 1 is selected from ISIS NO. 372844, 372922,372845, or 372923.

In certain embodiments, a target region is nucleotides 5588-5609 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 5588-5609 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 84, 85, 86, 87, 88, 89, 90, 91, or 92.In certain such embodiments, a short antisense compound targeted tonucleotides 5588-5609 of SEQ ID NO: 1 is selected from ISIS NO. 346601,346602, 346603, 346604, 346605, 346606, 346607, 346608, or 346609.

In certain embodiments, a target region is nucleotides 5924-5939 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 5924-5939 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 93 or 94. In certain such embodiments,a short antisense compound targeted to nucleotides 5924-5939 of SEQ IDNO: 1 is selected from ISIS NO. 372851 or 372929.

In certain embodiments, a target region is nucleotides 6664-6679 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 6664-6679 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 95 or 96. In certain such embodiments,a short antisense compound targeted to nucleotides 6664-6679 of SEQ IDNO: 1 is selected from ISIS NO. 372854 or 372932.

In certain embodiments, a target region is nucleotides 6908-6923 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 6908-6923 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 97 or 98. In certain such embodiments,a short antisense compound targeted to nucleotides 6908-6923 of SEQ IDNO: 1 is selected from ISIS NO. 372855 or 372933.

In certain embodiments, a target region is nucleotides 7190-7205 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 7190-7205 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 99 or 100. In certain such embodiments,a short antisense compound targeted to nucleotides 7190-7205 of SEQ IDNO: 1 is selected from ISIS NO. 372856 or 372934.

In certain embodiments, a target region is nucleotides 7817-7839 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 7817-7839 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 101, 102, 104, 105, 106, 107, 108, 109,110, or 111. In certain such embodiments, a short antisense compoundtargeted to nucleotides 7817-7839 of SEQ ID NO: 1 is selected from ISISNO. 372858, 372936, 346610, 346611, 346612, 346613, 346614, 346615,346616, 346617, or 346618.

In certain embodiments, a target region is nucleotides 7995-8010 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 7995-8010 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 112 or 113. In certain suchembodiments, a short antisense compound targeted to nucleotides7995-8010 of SEQ ID NO: 1 is selected from ISIS NO. 372859 or 372937.

In certain embodiments, a target region is nucleotides 8336-8356 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 8336-8356 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 114, 115, 116, or 117. In certain suchembodiments, a short antisense compound targeted to nucleotides8336-8356 of SEQ ID NO: 1 is selected from ISIS NO. 372861, 372939,372862, or 372940.

In certain embodiments, a target region is nucleotides 8539-8554 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 8539-8554 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 118 or 119. In certain suchembodiments, a short antisense compound targeted to nucleotides8539-8554 of SEQ ID NO: 1 is selected from ISIS NO. 372863 or 372941.

In certain embodiments, a target region is nucleotides 9344-9359 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 9344-9359 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 120 or 121. In certain suchembodiments, a short antisense compound targeted to nucleotides9344-9359 of SEQ ID NO: 1 is selected from ISIS NO. 372871 or 372949.

In certain embodiments, a target region is nucleotides 9515-9530 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 9515-9530 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 122 or 123. In certain suchembodiments, a short antisense compound targeted to nucleotides9515-9530 of SEQ ID NO: 1 is selected from ISIS NO. 372872 or 372950.

In certain embodiments, a target region is nucleotides 9794-9809 of SEQID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 9794-9809 of SEQ ID NO: 1 comprises a nucleotidesequence selected from SEQ ID NO 124 or 125. In certain suchembodiments, a short antisense compound targeted to nucleotides9794-9809 of SEQ ID NO: 1 is selected from ISIS NO. 372875 or 372953.

In certain embodiments, a target region is nucleotides 10157-10187 ofSEQ ID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 10157-10187 of SEQ ID NO: 1 comprises anucleotide sequence selected from SEQ ID NO 126, 127, 128, 129, 130,131, 132, or 133. In certain such embodiments, a short antisensecompound targeted to nucleotides 10157-10187 of SEQ ID NO: 1 is selectedfrom ISIS NO. 372877, 372955, 372878, 372956, 372879, 372957, 372880, or372958.

In certain embodiments, a target region is nucleotides 10838-10859 ofSEQ ID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 10838-10859 of SEQ ID NO: 1 comprises anucleotide sequence selected from SEQ ID NO 134, 135, 136, 137, 138,139, 140, 141, or

142. In certain such embodiments, a short antisense compound targeted tonucleotides 10838-10859 of SEQ ID NO: 1 is selected from ISIS NO.346619, 346620, 346621, 346622, 346623, 346624, 346625, 346626, or346627.

In certain embodiments, a target region is nucleotides 13689-13714 ofSEQ ID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 13689-13714 of SEQ ID NO: 1 comprises anucleotide sequence selected from SEQ ID NO 143, 144, 145, 146, 147, or148. In certain such embodiments, a short antisense compound targeted tonucleotides 13689-13714 of SEQ ID NO: 1 is selected from ISIS NO.372890, 372968, 372891, 372969, 372892, or 372970.

In certain embodiments, a target region is nucleotides 13907-13928 ofSEQ ID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 13907-13928 of SEQ ID NO: 1 comprises anucleotide sequence selected from SEQ ID NO 149, 150, 151, 152, 153,154, 155, 156, or 157. In certain such embodiments, a short antisensecompound targeted to nucleotides 13907-13928 of SEQ ID NO: 1 is selectedfrom ISIS NO. 346628, 346629, 346630, 346631, 346632, 346633, 346634,346635, or 346636.

In certain embodiments, a target region is nucleotides 13963-13984 ofSEQ ID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 13963-13984 of SEQ ID NO: 1 comprises anucleotide sequence selected from SEQ ID NO 158, 159, 160, 161, 162,163, 164, 165, or 166. In certain such embodiments, a short antisensecompound targeted to nucleotides 13963-13984 of SEQ ID NO: 1 is selectedfrom ISIS NO. 346637, 346638, 346639, 346640, 346641, 346642, 346643,346644, or 346645.

In certain embodiments, a target region is nucleotides 14051-14072 ofSEQ ID NO: 1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 14051-14072 of SEQ ID NO: 1 comprises anucleotide sequence selected from SEQ ID NO 167, 168, 169, 170, 171,172, 173, 174, or 175. In certain such embodiments, a short antisensecompound targeted to nucleotides 14051-14072 of SEQ ID NO: 1 is selectedfrom ISIS NO. 346646, 346647, 346648, 346649, 346650, 346651, 346652,346653, or 346654.

In certain embodiments, short antisense compounds targeted to an ApoBnucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 nucleotides in length. In certain embodiments,short antisense compounds targeted to an ApoB nucleic acid are 9 to 14nucleotides in length. In certain embodiments, short antisense compoundstargeted to an ApoB nucleic acid are 10 to 14 nucleotides in length. Incertain embodiments, such short antisense compounds are short antisenseoligonucleotides.

In certain embodiments, short antisense compounds targeted to an ApoBnucleic acid are short gapmers. In certain such embodiments, shortgapmers targeted to an ApoB nucleic acid comprise at least one highaffinity modification in one or more wings of the compound. In certainembodiments, short antisense compounds targeted to an ApoB nucleic acidcomprise 1 to 3 high-affinity modifications in each wing. In certainsuch embodiments, the nucleosides or nucleotides of the wing comprise a2′ modification. In certain such embodiments, the monomers of the wingare BNA's. In certain such embodiments, the monomers of the wing areselected from α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, β-D-Methyleneoxy(4′-CH₂—O-2′) BNA, Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, Aminooxy(4′-CH₂—O—N(R)-2′) BNA and Oxyamino (4′-CH₂—N(R)—O-2′) BNA. In certainembodiments, the monomers of a wing comprise a substituent at the 2′position selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m), and R_(n) is, independently, H or substituted or unsubstitutedC₁-C₁₀ alkyl. In certain embodiments, the monomers of a wing are 2′MOEnucleotides.

In certain embodiments, short antisense compounds targeted to an ApoBnucleic acid comprise a gap between the 5′ wing and the 3′ wing. Incertain embodiments the gap comprises five, six, seven, eight, nine,ten, eleven, twelve, thirteen, or fourteen monomers. In certainembodiments, the monomers of the gap are unmodifieddeoxyribonucleotides. In certain embodiments, the monomers of the gapare unmodified ribonucleotides. In certain embodiments, gapmodifications (if any) gap result in an antisense compound that, whenbound to its target nucleic acid, supports cleavage by an RNase,including, but not limited to, RNase H.

In certain embodiments, short antisense compounds targeted to an ApoBnucleic acid have uniform monomeric linkages. In certain suchembodiments, those linkages are all phosphorothioate linkages. Incertain embodiments, the linkages are all phosphodiester linkages. Incertain embodiments, short antisense compounds targeted to an ApoBnucleic acid have mixed backbones.

In certain embodiments, short antisense compounds targeted to an ApoBnucleic acid are 8 monomers in length. In certain embodiments, shortantisense compounds targeted to an ApoB nucleic acid are 9 monomers inlength. In certain embodiments, short antisense compounds targeted to anApoB nucleic acid are 10 monomers in length. In certain embodiments,short antisense compounds targeted to an ApoB nucleic acid are 11monomers in length. In certain embodiments, short antisense compoundstargeted to an ApoB nucleic acid are monomers in length. In certainembodiments, short antisense compounds targeted to an ApoB nucleic acidare 13 monomers in length. In certain embodiments, short antisensecompounds targeted to an ApoB nucleic acid are 14 monomers in length. Incertain embodiments, short antisense compounds targeted to an ApoBnucleic acid are 15 monomers in length. In certain embodiments, shortantisense compounds targeted to an ApoB nucleic acid are 16 monomers inlength. In certain embodiments, short antisense compounds targeted to anApoB nucleic acid comprise 9 to 15 monomers. In certain embodiments,short antisense compounds targeted to an ApoB nucleic acid comprise 10to 15 monomers. In certain embodiments, short antisense compoundstargeted to an ApoB nucleic acid comprise 12 to 14 monomers. In certainembodiments, short antisense compounds targeted to an ApoB nucleic acidcomprise 12 to 14 nucleotides or nucleosides.

In certain embodiments, the invention provides methods of modulatingexpression of ApoB. In certain embodiments, such methods comprise use ofone or more short antisense compound targeted to an ApoB nucleic acid,wherein the short antisense compound targeted to an ApoB nucleic acid isfrom about 8 to about 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linkedmonomers). One of ordinary skill in the art will appreciate that thiscomprehends methods of modulating expression of ApoB using one or moreshort antisense compounds targeted to an ApoB nucleic acid of 8, 9, 10,11, 12, 13, 14, 15 or 16 monomers.

In certain embodiments, methods of modulating ApoB comprise use of ashort antisense compound targeted to an ApoB nucleic acid that is 8monomers in length. In certain embodiments, methods of modulating ApoBcomprise use of a short antisense compound targeted to an ApoB nucleicacid that is 9 monomers in length. In certain embodiments, methods ofmodulating ApoB comprise use of a short antisense compound targeted toan ApoB nucleic acid that is 10 monomers in length. In certainembodiments, methods of modulating ApoB comprise use of a shortantisense compound targeted to an ApoB nucleic acid that is 11 monomersin length. In certain embodiments, methods of modulating ApoB compriseuse of a short antisense compound targeted to an ApoB nucleic acid thatis 12 monomers in length. In certain embodiments, methods of modulatingApoB comprise use of a short antisense compound targeted to an ApoBnucleic acid that is 13 monomers in length. In certain embodiments,methods of modulating ApoB comprise use of a short antisense compoundtargeted to an ApoB nucleic acid that is 14 monomers in length. Incertain embodiments, methods of modulating ApoB comprise use of a shortantisense compound targeted to an ApoB nucleic acid that is 15 monomersin length. In certain embodiments, methods of modulating ApoB compriseuse of a short antisense compound targeted to an ApoB nucleic acid thatis 16 monomers in length.

In certain embodiments, methods of modulating expression of ApoBcomprise use of a short antisense compound targeted to an ApoB nucleicacid comprising 9 to 15 monomers. In certain embodiments, methods ofmodulating expression of ApoB comprise use of a short antisense compoundtargeted to an ApoB nucleic acid comprising 10 to 15 monomers. Incertain embodiments, methods of modulating expression of ApoB compriseuse of a short antisense compound targeted to an ApoB nucleic acidcomprising 12 to 14 monomers. In certain embodiments, methods ofmodulating expression of ApoB comprise use of a short antisense compoundtargeted to an ApoB nucleic acid comprising 12 or 14 nucleotides ornucleosides.

In certain embodiments, short antisense compounds targeting a ApoBnucleic acid may have any one or more properties or characteristics ofthe short antisense compounds generally described herein. In certainembodiments, short antisense compounds targeting a ApoB nucleic acidhave a motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2,2-10-1-1, 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1,3-6-3 or 1-6-1, more preferably 1-10-1, 2-10-2, 3-10-3, and 1-9-2.

2. SGLT-2

Sodium dependent glucose transporter 2 (SGLT-2) is expressed in thekidney proximal tubule epithelial cells, and functions to reabsorbglucose preventing glucose loss in the urine. For the human genomeSGLT-2 is a member of an 11-membered family of sodium substrateco-transporters. Many of these family members share sequence homology,for example SGLT-1 shares about 59% sequence identity with SGLT-2 andabout 70% sequence identity with SGLT-3. SGLT-1 is a glucose transporterfound in the heart and the CNS. SGLT-3 is a glucose sensing sodiumchannel in the small intestine. The separate localization patterns forthese SGLTs is one point of distinction between the homologous familymembers. (Handlon, A. L., Expert Opin. Ther. Patents (2005)15(11):1532-1540; Kanai et al., J. Clin. Invest., 1994, 93, 397-404;Wells et al., Am. J. Physiol. Endocrinol. Metab., 1992, 263, F459-465).

Studies of human SGLT2 injected into Xenopus oocytes demonstrated thatthis protein mediates sodium-dependent transport of D-glucose and.alpha.-methyl-D-glucopyranoside (.alpha.-MeGlc; a glucose analog) witha Km value of 1.6 mM for .alpha.-MeGlc and a sodium to glucose couplingratio of 1:1 (Kanai et al., J. Clin. Invest., 1994, 93, 397-404; You etal., J. Biol. Chem., 1995, 270, 29365-29371). This transport activitywas suppressed by phlorizin, a plant glycoside that binds to the glucosesite of the SGLTs but is not transported and thus inhibits SGLT action(You et al., J. Biol. Chem., 1995, 270, 29365-29371).

Diabetes is a disorder characterized by hyperglycemia due to deficientinsulin action. Chronic hyperglycemia is a major risk factor fordiabetes-associated complications, including heart disease, retinopathy,nephropathy and neuropathy. As the kidneys play a major role in theregulation of plasma glucose levels, renal glucose transporters arebecoming attractive drug targets (Wright, Am. J. Physiol. RenalPhysiol., 2001, 280, F10-18). Diabetic nephropathy is the most commoncause of end-stage renal disease that develops in many patients withdiabetes. Glucotoxicity, which results from long-term hyperglycemia,induces tissue-dependent insulin resistance in diabetic patients (Nawanoet al., Am. J. Physiol. Endocrinol. Metab., 2000, 278, E535-543).

Definitions

“Sodium dependent glucose transporter 2” is the gene product or proteinof which expression is to be modulated by administration of a shortantisense compound. Sodium dependent glucose transporter 2 is generallyreferred to as SGLT2 but may also be referred to as SLC5A2;sodium-glucose transporter 2; sodium-glucose cotransporter, kidney lowaffinity; sodium-glucose cotransporter, renal; solute carrier family 5(sodium/glucose cotransporter), member 2; SL52.

“SGLT2 nucleic acid” means any nucleic acid encoding SGLT2. For example,in certain embodiments, a SGLT2 nucleic acid includes, withoutlimitation, a DNA sequence encoding SGLT2, an RNA sequence transcribedfrom DNA encoding SGLT2, and an mRNA sequence encoding SGLT2. “SGLT2mRNA” means an mRNA encoding a SGLT2 protein.

Therapeutic Indications

In certain embodiments, short antisense compounds are used to modulateexpression of SGLT-2 and related proteins. In certain embodiments, suchmodulation is accomplished by providing short antisense compounds thathybridize with one or more target nucleic acid molecules encodingSGLT-2, including, but is not limited to, SGLT2, SL52, SLC5A2,Sodium-Glucose Co-Transporter, Kidney Low Affinity Sodium-GlucoseCo-Transporter, Renal Sodium-Glucose Co-Transporter 2 and Solute CarrierFamily 5 Sodium/Glucose Co-Transporter Member 2. Also provided aremethods of treating metabolic and/or cardiovascular disease anddisorders as described herein. In particular embodiments, shortantisense compounds that inhibit the expression of SGLT2 are used inmethods of lowering blood glucose levels in an animal and methods ofdelaying or preventing the onset of type 2 diabetes. Such methodscomprise administering a therapeutically or prophylactically effectiveamount of one or more of the compounds of the invention to the animal,which may be in need of treatment. The one or more compounds can be ashort antisense compound targeting a nucleic acid encoding SGLT2.Provided herein are methods of enhancing inhibition of expression ofSGLT2 in kidney cells or kidney tissues, comprising contacting the cellsor tissues with one or more of the compounds of the invention, such asshort antisense compounds targeting a nucleic acid encoding SGLT2.

While certain compounds, compositions and methods have been describedwith specificity in accordance with certain embodiments, the followingexamples serve only to illustrate the compounds of the invention and arenot intended to limit the same.

In certain embodiments, short antisense compounds are chimericoligomeric compounds having mixed phosphorothioate and phosphodiesterbackbones. Certain mixed backbone short antisense compounds have acentral gap comprising at least 5 contiguous 2′-deoxy nucleosidesflanked by two wings each of which comprises at least one2′-O-methoxyethyl nucleoside. In certain embodiments, theinternucleoside linkages of the mixed backbone compounds arephosphorothioate linkages in the gap and phosphodiester linkages in thetwo wings. In certain embodiments, mixed backbone compounds havephosphorothioate linkages in the wings, except for one phosphodiesterlinkage at one or both of the extreme 5′ and 3′ ends of theoligonucleotide. In certain embodiments short antisense compoundstargeted to SGLT2 have a motif (wing-deoxy gap-wing) selected from3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 2-8-2, 1-9-2, 1-8-1, 3-6-3 or1-6-1. In certain embodiments short antisense compounds targeted toSGLT2 have a motif (wing-deoxy gap-wing) selected from 1-10-1, 1-10-2,2-8-2, 1-9-2, 1-8-1, 3-6-3 or 1-6-1.

In certain embodiments, short antisense compounds targeted to an SGLT2nucleic acid and having a mixed backbone are efficiently delivered tothe kidney. In certain embodiments, administration of short antisensecompounds targeted to an SGLT2 nucleic acid and having a mixed backboneresults in modulation of target gene expression in the kidney. Incertain such embodiments, there is little or no liver or kidneytoxicity. In certain embodiments, short antisense compounds targeted toan SGLT2 nucleic acid and having a mixed backbone are more potent forreducing SGLT-2 mRNA and have a faster onset compared with a shortantisense compound that does not have a mixed back-bone, but isotherwise identical. In certain such embodiments, such increase potencyand/or reduced toxicity is in mouse and/or rat. In certain suchembodiments, such increase potency and/or reduced toxicity is in ahuman.

By way of example, and only for illustrative purposes, ISIS 145733,which comprises uniform phosphorothioate linkages and ISIS 257016 whichcomprises phosphodiester linkage in the wings and phosphorothioatelinkages in the gap, are otherwise identical. Both comprise the sequenceGAAGTAGCCACCAACTGTGC (SEQ ID NO. 1572). Both of the oligonucleotidesfurther comprise a gap consisting of ten 2′-deoxynucleotides, flanked oneach side by five-nucleotide “2′-methoxyethyl (2′-MOE) nucleotides. Allcytidine residues are 5-methylcytidines. The mixed back-bone compound,ISIS 257016, was about 50 times more potent for reducing SGLT-2 mRNAcompared to the non-mixed parent compound, ISIS 145733 (see EXAMPLE 9).

Pharmacokinetic studies of certain mixed backbone compound ISIS 257016indicate that in certain embodiments, the compound acts as a prodrugthat is metabolized to a 12 nucleobase pharmacophore. Studies with ISIS370717, a 12 nucleobase short antisense compound corresponding to ISIS257016, show that the compound has a similar pharmacological profile toISIS 257016 but with a faster onset of action. ISIS 370717 is a 12nucleobase antisense oligonucleotide targeted to SGLT-2 comprising thesequence TAGCCACCAACT (SEQ ID NO. 1554), further comprising a gapconsisting of ten 2′-deoxynucleotides, flanked on both sides byone-nucleotide wings. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. All cytidine residues are 5-methylcytidines. Theinternucleoside linkages are phosphorothioate (P═S) throughout theoligonucleotide. The similarity in pharmacological activity of ISIS257016 and ISIS 370717 supports the pharmacokinetic studies indicatingISIS 257016 was a prodrug having a 12 nucleotide pharmacophore (seeEXAMPLE 10). Further, studies with stabilized (end-capped) versions ofISIS 257016 show dramatic loss of activity.

In certain embodiments, short antisense compounds comprising 2′ MOEmonomers in the wings are efficiently delivered to the kidney andtreatment with such compounds results in efficient modulation of targetgene expression in the kidney without liver or kidney toxicity. It isfurther shown herein that in certain embodiments, short antisensecompounds are more potent for reducing SGLT-2 mRNA and have a fasteronset compared with parent oligonucleotides targeted to SGLT-2 mRNA inmouse and rat. 2′ MOE gap shortmers are shown herein to improve potencyand bioavailability over parent compounds.

By way of example, and only for illustrative purposes studies with ISIS370717 reveal significantly higher accumulation of the short antisensecompound in the kidney tissue (approximately 500 micro grams per gram oftissue) compared to the longer parent. Moreover, SGLT-2 mRNA was reducedby more than 80% over the controls (see EXAMPLE 11). ISIS 3707171-10-1gapmer was used as a template to make sequence related oligos withvarying motifs. Studies evaluating wing, gap and total length variationsaround the ISIS 370717 12 mer oligonucleotide can be seen in EXAMPLE 12.Certain motifs evaluated included 1-10-1, 2-8-2, 1-8-1, 3-6-3, and 1-6-1(see Table 60 in EXAMPLE 12). The compounds were analyzed for theireffect on SGLT2 mRNA levels. All the motifs inhibited the expression ofSGLT2 in vivo in a dose-dependent manner. The 1-10-1, 2-8-2 and 1-8-1gapmers were found to be particularly potent. SGLT-2 mRNA was reduced bymore than 80% over the controls using these motifs.

In certain embodiments, the invention provides short antisense compoundstargeted to an SGLT2 nucleic acid and having a motif selected from:1-10-1 and 1-10-2 MOE gapmer. (see Table 62 in EXAMPLE 13). Certain suchcompounds were analyzed for their effect on rat SGLT2 mRNA. Results inTable 63 illustrate that both the 1-10-1 and 1-10-2 MOE gapmers inhibitthe expression of SGLT2 in vivo in a dose-dependent manner and over 80%reduction of SGLT-2 mRNA could be achieved.

Certain additional 1-10-1 and 2-8-2 MOE gapmers were evaluated in bothmouse and rat in vivo models (see, e.g., EXAMPLE 14 and 15). Greaterthan 80% reduction in SGLT-2 mRNA was achieved with many of the 1-10-1and 2-8-2 MOE gapmers at relatively low concentrations of oligo and inthe absence of any toxicity effects.

In another non-limiting example, the effect of ISIS 388625 on dog SGLT2mRNA levels was also analyzed. Dog studies illustrate that greater than80% inhibition of the expression of SGLT2 can be achieved at a 1mg/kg/wk dose. Even greater inhibition can be achieved at slightlyhigher doses. Administration of ISIS 388625 in dog was also shown toimproved glucose tolerance. Peak plasma glucose levels were decreased byover 50% on average and the subsequent drop in glucose was lessenedcompared to saline controls in a standard glucose tolerance test (SeeEXAMPLE 17). Also, in a rat model of diabetes, short antisense compoundswere shown to significantly decrease plasma glucose levels and HbA1Cover time compared to PBS and control treated animals (See Example 16).

The animals in all studies were further evaluated for toxicity. Forexample, total body weight, liver, spleen and kidney weight wereevaluated. Significant changes in spleen, liver or body weight canindicate that a particular compound causes toxic effects. All changeswere found to be within the margin of error. No significant changes inbody weight were observed during the treatment or at study termination.No significant changes in liver or spleen weights were observed.

Certain Short Antisense Compounds Targeted to an SGLT2 Nucleic Acid

In certain embodiments, short antisense compounds are targeted to anSGLT2 nucleic acid having the sequence of GENBANK® Accession No.NM_(—)003041.1, incorporated herein as SEQ ID NO: 2. In certain suchembodiments, a short antisense compound targeted to SEQ ID NO: 3 is atleast 90% complementary to SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to SEQ ID NO: 3 is at least 95%complementary to SEQ ID NO: 3. In certain such embodiments, a shortantisense compound targeted to SEQ ID NO: 3 is 100% complementary to SEQID NO: 1. In certain embodiments, a short antisense compound targeted toSEQ ID NO: 3 comprises a nucleotide sequence selected from thenucleotide sequences set forth in Table 4 and 5.

The nucleotide sequence set forth in each SEQ ID NO set forth in Tables4 and 5 is independent of any modification to a sugar moiety, amonomeric linkage, or a nucleobase. As such, short antisense compoundsdefined by a SEQ ID NO may comprise, independently, one or moremodifications to a sugar moiety, an internucleoside linkage, or anucleobase. Antisense compounds described by Isis Number (Isis NO.)indicate a combination of nucleobase sequence and one or moremodifications to a sugar moiety, an internucleoside linkage, or anucleobase.

Tables 4 and 5 illustrate examples of short antisense compounds targetedto SEQ ID NO: 3. Table 4 illustrates short antisense compounds that are100% complementary to SEQ ID NO: 3. Table 5 illustrates short antisensecompounds that have one or two mismatches with respect to SEQ ID NO: 3.The column labeled ‘gapmer motif’ indicates the wing-gap-wing motif ofeach short antisense compounds. The gap segment comprises2′-deoxynucleotides and each nucleotide of each wing segment comprises a2′-modified sugar. The particular 2′-modified sugar is also indicated inthe ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flankedby wing segments of two nucleotides, where the nucleotides of the wingsegments are 2′-MOE nucleotides. Internucleoside linkages arephosphorothioate. The short antisense compounds comprise5-methylcytidine in place of unmodified cytosine, unless “unmodifiedcytosine” is listed in the gapmer motif column, in which case theindicated cytosines are unmodified cytosines. For example, “5-mC in gaponly” indicates that the gap segment has 5-methylcytosines, while thewing segments have unmodified cytosines.

TABLE 4 Short Antisense Compounds Targeted to SEQ ID NO: 3 5′ 3′ SEQISIS Target Target Sequence ID No Site Site (5′-3′) Gapmer Motif NO379684 84 95 TGTCAGCAGGAT 1-10-1 MOE 214 405193 113 124 CAGCAGGAAATA2-8-2 MOE 215 405194 114 125 CCAGCAGGAAAT 2-8-2 MOE 216 405195 115 126ACCAGCAGGAAA 2-8-2 MOE 217 405196 116 127 GACCAGCAGGAA 2-8-2 MOE 218405197 117 128 TGACCAGCAGGA 2-8-2 MOE 219 379685 117 128 TGACCAGCAGGA1-10-1 MOE 219 405198 118 129 ATGACCAGCAGG 2-8-2 MOE 221 405199 119 130AATGACCAGCAG 2-8-2 MOE 222 405200 120 131 CAATGACCAGCA 2-8-2 MOE 223405201 121 132 CCAATGACCAGC 2-8-2 MOE 224 379686 135 146 ACCACAAGCCAA1-10-1 MOE 225 379711 172 183 TAGCCGCCCACA 1-10-1 MOE 226 388628 172 183TAGCCGCCCACA 2-8-2 MOE 226 405202 207 218 CCGGCCACCACA 2-8-2 MOE 228405203 208 219 ACCGGCCACCAC 2-8-2 MOE 229 405204 236 247 GATGTTGCTGGC2-8-2 MOE 230 379687 236 247 GATGTTGCTGGC 1-10-1 MOE 230 405205 237 248CGATGTTGCTGG 2-8-2 MOE 232 405206 238 249 CCGATGTTGCTG 2-8-2 MOE 233405207 239 250 GCCGATGTTGCT 2-8-2 MOE 234 405208 240 251 TGCCGATGTTGC2-8-2 MOE 235 405209 241 252 CTGCCGATGTTG 2-8-2 MOE 236 405210 260 271CAGGCCCACAAA 2-8-2 MOE 237 405211 261 272 CCAGGCCCACAA 2-8-2 MOE 238405212 262 273 GCCAGGCCCACA 2-8-2 MOE 239 379688 288 299 CCAAGCCACTTG1-10-1 MOE 240 379689 318 329 AGAGCGCATTCC 1-10-1 MOE 241 379690 435 446ACAGGTAGAGGC 1-10-1 MOE 242 405248 474 485 AGATCTTGGTGA 2-8-2 MOE 243379691 474 485 AGATCTTGGTGA 1-10-1 MOE 243 382676 527 539 TGTTCCAGCCCAG1-10-2 MOE 245 388625 528 539 TGTTCCAGCCCA 2-8-2 MOE 246 389780 528 539TGTTCCAGCCCA 1-9-2 MOE 246 379692 528 539 TGTTCCAGCCCA 1-10-1 MOE 246392170 528 539 TGTTCCAGCCCA 1-10-1 246 Methyleneoxy BNA 392173 528 539TGTTCCAGCCCA 2-8-2 246 Methyleneoxy BNA 405213 529 540 ATGTTCCAGCCC2-8-2 MOE 251 405214 564 575 TGGTGATGCCCA 2-8-2 MOE 252 405215 565 576ATGGTGATGCCC 2-8-2 MOE 253 405216 566 577 CATGGTGATGCC 2-8-2 MOE 254379693 566 577 CATGGTGATGCC 1-10-1 MOE 254 405217 567 578 TCATGGTGATGC2-8-2 MOE 256 405218 568 579 ATCATGGTGATG 2-8-2 MOE 257 405219 587 598CCCTCCTGTCAC 2-8-2 MOE 258 405220 588 599 GCCCTCCTGTCA 2-8-2 MOE 259405221 589 600 AGCCCTCCTGTC 2-8-2 MOE 260 405222 590 601 CAGCCCTCCTGT2-8-2 MOE 261 405223 591 602 CCAGCCCTCCTG 2-8-2 MOE 262 405224 592 603GCCAGCCCTCCT 2-8-2 MOE 263 379694 629 640 GACGAAGGTCTG 1-10-1 MOE 264405225 707 718 GTATTTGTCGAA 2-8-2 MOE 265 379695 737 748 GGACACCGTCAG1-10-1 MOE 266 379696 974 985 CAGCTTCAGGTA 1-10-1 MOE 267 405226 9981009 CATGACCATGAG 2-8-2 MOE 268 405227 999 1010 GCATGACCATGA 2-8-2 MOE269 405228 1000 1011 GGCATGACCATG 2-8-2 MOE 270 405229 1001 1012TGGCATGACCAT 2-8-2 MOE 271 405230 1002 1013 CTGGCATGACCA 2-8-2 MOE 272379697 1002 1013 CTGGCATGACCA 1-10-1 MOE 272 405231 1003 1014CCTGGCATGACC 2-8-2 MOE 274 379698 1091 1102 GCAGCCCACCTC 1-10-1 MOE 275405232 1092 1103 AGCAGCCCACCT 2-8-2 MOE 276 405233 1093 1104GAGCAGCCCACC 2-8-2 MOE 277 405234 1130 1141 CATGAGCTTCAC 2-8-2 MOE 278405235 1131 1142 GCATGAGCTTCA 2-8-2 MOE 279 382677 1131 1143GGCATGAGCTTCA 1-10-2 MOE 280 388626 1132 1143 GGCATGAGCTTC 2-8-2 MOE 281379699 1132 1143 GGCATGAGCTTC 1-10-1 MOE 281 405236 1133 1144GGGCATGAGCTT 2-8-2 MOE 283 405237 1157 1168 CAGCATGAGTCC 2-8-2 MOE 284405238 1158 1169 CCAGCATGAGTC 2-8-2 MOE 285 379700 1158 1169CCAGCATGAGTC 1-10-1 MOE 285 405239 1159 1170 GCCAGCATGAGT 2-8-2 MOE 287379701 1230 1241 CCATGGTGAAGA 1-10-1 MOE 288 405240 1542 1553CACAGCTGCCCG 2-8-2 MOE 289 405241 1543 1554 ACACAGCTGCCC 2-8-2 MOE 290405242 1544 1555 CACACAGCTGCC 2-8-2 MOE 291 382678 1544 1556GCACACAGCTGCC 1-10-2 MOE 292 388627 1545 1556 GCACACAGCTGC 2-8-2 MOE 293379702 1545 1556 GCACACAGCTGC 1-10-1 MOE 293 379703 1701 1712GCCGGAGACTGA 1-10-1 MOE 295 405243 1976 1987 ATTGAGGTTGAC 2-8-2 MOE 296405244 1977 1988 CATTGAGGTTGA 2-8-2 MOE 297 405245 1978 1989GCATTGAGGTTG 2-8-2 MOE 298 405246 1979 1990 GGCATTGAGGTT 2-8-2 MOE 299405247 1980 1991 GGGCATTGAGGT 2-8-2 MOE 300

TABLE 5 Short antisense compounds targeted to SEQ ID NO: 3 and having 1or 2 mismatches 5′ 3′ SEQ ISIS Target Target Sequence ID No Site Site(5′-3′) Gapmer Motif NO 405200 96 107 CAATGACCAGCA 2-8-2 MOE 223 405215382 393 ATGGTGATGCCC 2-8-2 MOE 253 405216 383 394 CATGGTGATGCC 2-8-2 MOE254 379693 383 394 CATGGTGATGCC 1-10-1 MOE 254 379701 471 482CCATGGTGAAGA 1-10-1 MOE 288 405218 472 483 ATCATGGTGATG 2-8-2 MOE 257405246 536 547 GGCATTGAGGTT 2-8-2 MOE 299 405248 570 581 AGATCTTGGTGA2-8-2 MOE 243 379691 570 581 AGATCTTGGTGA 1-10-1 MOE 243 379698 683 694GCAGCCCACCTC 1-10-1 MOE 275 405232 684 695 AGCAGCCCACCT 2-8-2 MOE 276379711 685 696 TAGCCGCCCACA 1-10-1 MOE 226 388628 685 696 TAGCCGCCCACA2-8-2 MOE 226 379698 950 961 GCAGCCCACCTC 1-10-1 MOE 275 405232 951 962AGCAGCCCACCT 2-8-2 MOE 276 405235 978 989 GCATGAGCTTCA 2-8-2 MOE 279382677 978 990 GGCATGAGCTTCA 1-10-2 MOE 280 388626 979 990 GGCATGAGCTTC2-8-2 MOE 281 379699 979 990 GGCATGAGCTTC 1-10-1 MOE 281 405236 980 991GGGCATGAGCTT 2-8-2 MOE 283 379698 1043 1054 GCAGCCCACCTC 1-10-1 MOE 275405239 1171 1182 GCCAGCATGAGT 2-8-2 MOE 287 405209 1213 1224CTGCCGATGTTG 2-8-2 MOE 236 405233 1364 1375 GAGCAGCCCACC 2-8-2 MOE 277405240 1366 1377 CACAGCTGCCCG 2-8-2 MOE 289 405211 1500 1511CCAGGCCCACAA 2-8-2 MOE 238 405212 1501 1512 GCCAGGCCCACA 2-8-2 MOE 239379695 1643 1654 GGACACCGTCAG 1-10-1 MOE 266 379698 1875 1886GCAGCCCACCTC 1-10-1 MOE 275 405239 1993 2004 GCCAGCATGAGT 2-8-2 MOE 287405211 2210 2221 CCAGGCCCACAA 2-8-2 MOE 238 405212 2211 2222GCCAGGCCCACA 2-8-2 MOE 239

In certain embodiments, a target region is nucleotides 85-184 of SEQ IDNO: 3. In certain embodiments, a short antisense compound is targeted tonucleotides 85-184 of SEQ ID NO: 3. In certain such embodiments, a shortantisense compound targeted to nucleotides 85-184 comprises a nucleotidesequence selected from SEQ ID NO 214, 215, 216, 217, 218, 219, 221, 222,223, 224, 225, or 227. In certain such embodiments, a short antisensecompound targeted to nucleotides 85-184 of SEQ ID NO: 3 is selected fromIsis No 379684, 405193, 405194, 405195, 405196, 405197, 379685, 405198,405199, 405200, 405201, 379686, 379711 or 388628.

In certain embodiments, a target region is nucleotides 113-132 of SEQ IDNO: 3. In certain embodiments, a short antisense compound is targeted tonucleotides 113-132 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 113-132 comprises anucleotide sequence selected from SEQ ID NO 215, 216, 217, 218, 219,221, 222, 223, or 224. In certain such embodiments, a short antisensecompound targeted to nucleotides 113-132 of SEQ ID NO: 3 is selectedfrom Isis No 405193, 405194, 405195, 405196, 405197, 379685, 405198,405199, 405200, or 405201.

In certain embodiments, a target region is nucleotides 207-329 of SEQ IDNO: 3. In certain embodiments, a short antisense compound is targeted tonucleotides 207-329 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 207-329 comprises anucleotide sequence selected from SEQ ID NO 228, 229, 230, 232, 233,234, 235, 236, 237, 238, 239, 240, or 241. In certain such embodiments,a short antisense compound targeted to nucleotides 207-329 of SEQ ID NO:3 is selected from Isis No 405202, 405203, 405204, 379687, 405205,405206, 405207, 405208, 405209, 405210, 405211, 405212, 379688, or379689.

In certain embodiments, a target region is nucleotides 207-273 of SEQ IDNO: 3. In certain embodiments, a short antisense compound is targeted tonucleotides 207-273 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 207-273 comprises anucleotide sequence selected from SEQ ID NO 228, 229, 230, 232, 233,234, 235, 236, 237, 238, or 239. In certain such embodiments, a shortantisense compound targeted to nucleotides 207-273 of SEQ ID NO: 3 isselected from Isis No 405202, 405203, 405204, 379687, 405205, 405206,405207, 405208, 405209, 405210, 405211, or 405212.

In certain embodiments, a target region is nucleotides 207-219 of SEQ IDNO: 3. In certain embodiments, a short antisense compound is targeted tonucleotides 207-219 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 207-219 comprises anucleotide sequence selected from SEQ ID NO 228 or 229. In certain suchembodiments, a short antisense compound targeted to nucleotides 207-219of SEQ ID NO: 3 is selected from Isis NO. 405202 or 405203.

In certain embodiments, a target region is nucleotides 236-252 of SEQ IDNO: 3. In certain embodiments, a short antisense compound is targeted tonucleotides 236-252 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 236-252 comprises anucleotide sequence selected from SEQ ID NO 230, 232, 233, 234, 235, or236. In certain such embodiments, a short antisense compound targeted tonucleotides 236-252 of SEQ ID NO: 3 is selected from Isis NO. 405204,379687, 405205, 405206, 405207, 405208, or 405209.

In certain embodiments, a target region is nucleotides 260-273 of SEQ IDNO: 3. In certain embodiments, a short antisense compound is targeted tonucleotides 260-273 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 260-273 comprises anucleotide sequence selected from SEQ ID NO 237, 238, or 239. In certainsuch embodiments, a short antisense compound targeted to nucleotides260-273 of SEQ ID NO: 3 is selected from Isis NO. 405210, 405211, or405212.

In certain embodiments, a target region is nucleotides 435-640 of SEQ IDNO: 3. In certain embodiments, a short antisense compound is targeted tonucleotides 435-640 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 435-640 comprises anucleotide sequence selected from SEQ ID NO 242, 243, 245, 246, 251,252, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, or 264. Incertain such embodiments, a short antisense compound targeted tonucleotides 435-640 of SEQ ID NO: 3 is selected from Isis NO. 379690,405248, 379691, 389780, 379692, 382676, 388625, 392170, 392173, 405213,405214, 405215, 405216, 379693, 405217, 405218, 405219, 405220, 405221,405222, 405223, 405224, or 379694.

In certain embodiments, a target region is nucleotides 527-540 of SEQ IDNO: 3. In certain embodiments, a short antisense compound is targeted tonucleotides 527-540 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 527-540 comprises anucleotide sequence selected from SEQ ID NO 245, 246, or 251. In certainsuch embodiments, a short antisense compound targeted to nucleotides527-540 of SEQ ID NO: 3 is selected from Isis NO. 389780, 379692,382676, 388626, 392170, 392173, or 405213.

In certain embodiments, a target region is nucleotides 564-603 of SEQ IDNO: 3. In certain embodiments, a short antisense compound is targeted tonucleotides 564-603 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 564-603 comprises anucleotide sequence selected from SEQ ID NO 252, 253, 254, 256, 257,258, 259, 260, 261, 262, or 263. In certain such embodiments, a shortantisense compound targeted to nucleotides 564-603 of SEQ ID NO: 3 isselected from Isis NO. 405214, 405215, 405216, 379693, 405217, 405218,405219, 405220, 405221, 405222, 405223, or 405224.

In certain embodiments, a target region is nucleotides 564-579 of SEQ IDNO: 3. In certain embodiments, a short antisense compound is targeted tonucleotides 564-579 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 564-579 comprises anucleotide sequence selected from SEQ ID NO 252, 253, 254, 256, or 257.In certain such embodiments, a short antisense compound targeted tonucleotides 564-579 of SEQ ID NO: 3 is selected from Isis NO. 405214,405215, 405216, 379693, 405217, or 405218.

In certain embodiments, a target region is nucleotides 587-603 of SEQ IDNO: 3. In certain embodiments, a short antisense compound is targeted tonucleotides 587-603 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 587-603 comprises anucleotide sequence selected from SEQ ID NO 258, 259, 260, 261, 262, or263. In certain such embodiments, a short antisense compound targeted tonucleotides 587-603 of SEQ ID NO: 3 is selected from Isis NO. 405219,405220, 405221, 405222, 405223, or 405224.

In certain embodiments, a target region is nucleotides 974-1014 of SEQID NO: 3. In certain embodiments, a short antisense compound is targetedto nucleotides 974-1014 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 974-1014 comprises anucleotide sequence selected from SEQ ID NO 267, 268, 269, 270, 271,272, or 274. In certain such embodiments, a short antisense compoundtargeted to nucleotides 974-1014 of SEQ ID NO: 3 is selected from IsisNO. 379696, 405226, 405227, 405228, 405229, 405230, 379697, or 405231.

In certain embodiments, a target region is nucleotides 998-1014 of SEQID NO: 3. In certain embodiments, a short antisense compound is targetedto nucleotides 998-1014 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 998-1014 comprises anucleotide sequence selected from SEQ ID NO 268, 269, 270, 271, 272, or274. In certain such embodiments, a short antisense compound targeted tonucleotides 998-1014 of SEQ ID NO: 3 is selected from Isis NO. 405226,405227, 405228, 405229, 405230, 379697, or 405231.

In certain embodiments, a target region is nucleotides 1091-1170 of SEQID NO: 3. In certain embodiments, a short antisense compound is targetedto nucleotides 1091-1170 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 1091-1170 comprises anucleotide sequence selected from SEQ ID NO 275, 276, 277, 278, 279,280, 281, 283, 284, 285, 286, or 287. In certain such embodiments, ashort antisense compound targeted to nucleotides 1091-1170 of SEQ ID NO:3 is selected from Isis NO. 379698, 405232, 405233, 405234, 405235,388626, 379699, 382677, 405236, 405237, 405238, 379700, or 405239.

In certain embodiments, a target region is nucleotides 1091-1104 of SEQID NO: 3. In certain embodiments, a short antisense compound is targetedto nucleotides 1091-1104 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 1091-1104 comprises anucleotide sequence selected from SEQ ID NO 275, 276, or 277. In certainsuch embodiments, an short antisense compound targeted to nucleotides1091-1104 of SEQ ID NO: 3 is selected from Isis NO. 379698, 405232, or405233.

In certain embodiments, a target region is nucleotides 1130-1144 of SEQID NO: 3. In certain embodiments, a short antisense compound is targetedto nucleotides 1130-1144 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 1130-1144 comprises anucleotide sequence selected from SEQ ID NO 278, 279, 280, 281, or 283.In certain such embodiments, a short antisense compound targeted tonucleotides 1130-1144 of SEQ ID NO: 3 is selected from Isis NO. 405234,405235, 388626, 379699, 382677, or 405236.

In certain embodiments, a target region is nucleotides 1157-1170 of SEQID NO: 3. In certain embodiments, a short antisense compound is targetedto nucleotides 1157-1170 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 1157-1170 comprises anucleotide sequence selected from SEQ ID NO 284, 285, or 287. In certainsuch embodiments, a short antisense compound targeted to nucleotides1157-1170 of SEQ ID NO: 3 is selected from Isis NO. 405237, 405238,379700, or 405239.

In certain embodiments, a target region is nucleotides 1542-1556 of SEQID NO: 3. In certain embodiments, a short antisense compound is targetedto nucleotides 1542-1556 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 1542-1556 comprises anucleotide sequence selected from SEQ ID NO 289, 290, 291, 292, or 293.In certain such embodiments, a short antisense compound targeted tonucleotides 1542-1556 of SEQ ID NO: 3 is selected from Isis NO. 405240,405241, 405242, 388629, 379702, or 382678.

In certain embodiments, a target region is nucleotides 1976-1991 of SEQID NO: 3. In certain embodiments, a short antisense compound is targetedto nucleotides 1976-1991 of SEQ ID NO: 3. In certain such embodiments, ashort antisense compound targeted to nucleotides 1976-1991 comprises anucleotide sequence selected from SEQ ID NO 296, 297, 298, 299, or 300.In certain such embodiments, a short antisense compound targeted tonucleotides 1976-1991 of SEQ ID NO: 3 is selected from Isis NO. 405243,405244, 405245, 405246, or 405247.

In certain embodiments, short antisense compounds targeted to an SGLT2nucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 nucleotides in length. In certain embodiments,short antisense compounds targeted to an SGLT2 nucleic acid are 9 to 14nucleotides in length. In certain embodiments, short antisense compoundstargeted to an SGLT2 nucleic acid are 10 to 14 nucleotides in length. Incertain embodiments, such short antisense compounds are short antisenseoligonucleotides.

In certain embodiments, short antisense compounds targeted to an SGLT2nucleic acid are short gapmers. In certain such embodiments, shortgapmers targeted to an SGLT2 nucleic acid comprise at least one highaffinity modification in one or more wings of the compound. In certainembodiments, short antisense compounds targeted to an SGLT2 nucleic acidcomprise 1 to 3 high-affinity modifications in each wing. In certainsuch embodiments, the nucleosides or nucleotides of the wing comprise a2′ modification. In certain such embodiments, the monomers of the wingare BNA's. In certain such embodiments, the monomers of the wing areselected from α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, β-D-Methyleneoxy(4′-CH₂—O-2′) BNA, Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, Aminooxy(4′-CH₂—O—N(R)-2′) BNA and Oxyamino (4′-CH₂—N(R)—O-2′) BNA. In certainembodiments, the monomers of a wing comprise a substituent at the 2′position selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H or substituted or unsubstitutedC₁-C₁₀ alkyl. In certain embodiments, the monomers of a wing are 2′MOEnucleotides.

In certain embodiments, short antisense compounds targeted to an SGLT2nucleic acid comprise a gap between the 5′ wing and the 3′ wing. Incertain embodiments the gap comprises five, six, seven, eight, nine,ten, eleven, twelve, thirteen, or fourteen monomers. In certainembodiments, the monomers of the gap are unmodifieddeoxyribonucleotides. In certain embodiments, the monomers of the gapare unmodified ribonucleotides. In certain embodiments, gapmodifications (if any) gap result in an antisense compound that, whenbound to its target nucleic acid, supports cleavage by an RNase,including, but not limited to, RNase H.

In certain embodiments, short antisense compounds targeted to an SGLT2nucleic acid have uniform monomeric linkages. In certain suchembodiments, those linkages are all phosphorothioate linkages. Incertain embodiments, the linkages are all phosphodiester linkages. Incertain embodiments, short antisense compounds targeted to an SGLT2nucleic acid have mixed backbones.

In certain embodiments, short antisense compounds targeted to an SGLT2nucleic acid are 8 monomers in length. In certain embodiments, shortantisense compounds targeted to an SGLT2 nucleic acid are 9 monomers inlength. In certain embodiments, short antisense compounds targeted to anSGLT2 nucleic acid are 10 monomers in length. In certain embodiments,short antisense compounds targeted to an SGLT2 nucleic acid are 11monomers in length. In certain embodiments, short antisense compoundstargeted to an SGLT2 nucleic acid are monomers in length. In certainembodiments, short antisense compounds targeted to an SGLT2 nucleic acidare 13 monomers in length. In certain embodiments, short antisensecompounds targeted to an SGLT2 nucleic acid are 14 monomers in length.In certain embodiments, short antisense compounds targeted to an SGLT2nucleic acid are 15 monomers in length. In certain embodiments, shortantisense compounds targeted to an SGLT2 nucleic acid are 16 monomers inlength. In certain embodiments, short antisense compounds targeted to anSGLT2 nucleic acid comprise 9 to 15 monomers. In certain embodiments,short antisense compounds targeted to an SGLT2 nucleic acid comprise 10to 15 monomers. In certain embodiments, short antisense compoundstargeted to an SGLT2 nucleic acid comprise 12 to 14 monomers. In certainembodiments, short antisense compounds targeted to an SGLT2 nucleic acidcomprise 12 to 14 nucleotides or nucleosides.

In certain embodiments, the invention provides methods of modulatingexpression of SGLT2. In certain embodiments, such methods comprise useof one or more short antisense compound targeted to an SGLT2 nucleicacid, wherein the short antisense compound targeted to an SGLT2 nucleicacid is from about 8 to about 16, preferably 9 to 15, more preferably 9to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16linked monomers). One of ordinary skill in the art will appreciate thatthis comprehends methods of modulating expression of SGLT2 using one ormore short antisense compounds targeted to an SGLT2 nucleic acid of 8,9, 10, 11, 12, 13, 14, 15 or 16 monomers.

In certain embodiments, methods of modulating SGLT2 comprise use of ashort antisense compound targeted to an SGLT2 nucleic acid that is 8monomers in length. In certain embodiments, methods of modulating SGLT2comprise use of a short antisense compound targeted to an SGLT2 nucleicacid that is 9 monomers in length. In certain embodiments, methods ofmodulating SGLT2 comprise use of a short antisense compound targeted toan SGLT2 nucleic acid that is 10 monomers in length. In certainembodiments, methods of modulating SGLT2 comprise use of a shortantisense compound targeted to an SGLT2 nucleic acid that is 11 monomersin length. In certain embodiments, methods of modulating SGLT2 compriseuse of a short antisense compound targeted to an SGLT2 nucleic acid thatis 12 monomers in length. In certain embodiments, methods of modulatingSGLT2 comprise use of a short antisense compound targeted to an SGLT2nucleic acid that is 13 monomers in length. In certain embodiments,methods of modulating SGLT2 comprise use of a short antisense compoundtargeted to an SGLT2 nucleic acid that is 14 monomers in length. Incertain embodiments, methods of modulating SGLT2 comprise use of a shortantisense compound targeted to an SGLT2 nucleic acid that is 15 monomersin length. In certain embodiments, methods of modulating SGLT2 compriseuse of a short antisense compound targeted to an SGLT2 nucleic acid thatis 16 monomers in length.

In certain embodiments, methods of modulating expression of SGLT2comprise use of a short antisense compound targeted to an SGLT2 nucleicacid comprising 9 to 15 monomers. In certain embodiments, methods ofmodulating expression of SGLT2 comprise use of a short antisensecompound targeted to an SGLT2 nucleic acid comprising 10 to 15 monomers.In certain embodiments, methods of modulating expression of SGLT2comprise use of a short antisense compound targeted to an SGLT2 nucleicacid comprising 12 to 14 monomers. In certain embodiments, methods ofmodulating expression of SGLT2 comprise use of a short antisensecompound targeted to an SGLT2 nucleic acid comprising 12 or 14nucleotides or nucleosides.

3. PCSK9

In individuals with autosomal dominant hypercholesterolemia (ADH),elevated LDL-C levels have been linked to mutations in the genesencoding LDL-receptor (LDL-R), apolipoprotein B (apoB), or proproteinconvertase subtilisin/kexin type 9 (PCSK9) (Abifadel et al., Nat.Genet., 2003, 34:154-156). PCSK9 was identified as a third locusassociated with ADH when gain-of-function mutations in PCSK9 were foundto be linked to elevated LDL-C levels. ApoB participates in theintracellular assembly and secretion of triglyceride-rich lipoproteinsand is a ligand for the LDL-R. PCSK9 is proposed to reduce LDL-Rexpression levels in the liver. Reduced LDL-R expression results inreduced hepatic uptake of circulating ApoB-containing lipoproteins,which in turn leads to elevated cholesterol.

Definitions

“PCSK9” is the gene product or protein of which expression is to bemodulated by administration of a short antisense compound.

“PCSK9 nucleic acid” means any nucleic acid encoding PCSK9. For example,in certain embodiments, a PCSK9 nucleic acid includes, withoutlimitation, a DNA sequence encoding PCSK9, an RNA sequence transcribedfrom DNA encoding PCSK9, and an mRNA sequence encoding PCSK9.

“PCSK9 mRNA” means an mRNA encoding PCSK9.

PCSK9 Therapeutic Indications

In certain embodiments, the invention provides methods of modulating theexpression of PCSK9 in an individual comprising administering a shortantisense compound targeted to a PCSK9 nucleic acid. In certainembodiments, the invention provides methods of treating an individualcomprising administering one or more pharmaceutical compositions of thepresent invention. In certain embodiments, the individual hashypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk ofdeveloping atherosclerosis, coronary heart disease, a history ofcoronary heart disease, early onset coronary heart disease, one or morerisk factors for coronary heart disease, type II diabetes, type IIdiabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia,hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholicsteatohepatitis, or non-alcoholic fatty liver disease.

Guidelines for lipid-lowering therapy were established in 2001 by AdultTreatment Panel III (ATP III) of the National Cholesterol EducationProgram (NCEP), and updated in 2004 (Grundy et al., Circulation, 2004,110, 227-239). The guidelines include obtaining a complete lipoproteinprofile, typically after a 9 to 12 hour fast, for determination ofLDL-C, total cholesterol, and HDL-C levels. According to the mostrecently established guidelines, LDL-C levels of 130-159 mg/dL, 160-189mg/dL, and greater than or equal to 190 mg/dL are considered borderlinehigh, high, and very high, respectively. Total cholesterol levels of200-239 and greater than or equal to 240 mg/dL are considered borderlinehigh and high, respectively. HDL-C levels of less than 40 mg/dL areconsidered low.

In certain embodiments, the individual has been identified as in need oflipid-lowering therapy. In certain such embodiments, the individual hasbeen identified as in need of lipid-lowering therapy according to theguidelines established in 2001 by Adult Treatment Panel III (ATP III) ofthe National Cholesterol Education Program (NCEP), and updated in 2004(Grundy et al., Circulation, 2004, 110, 227-239). In certain suchembodiments, the individual in need of lipid-lowering therapy has LDL-Cabove 190 mg/dL. In certain such embodiments, the individual in need oflipid-lowering therapy has LDL-C above 160 mg/dL. In certain suchembodiments, the individual in need of lipid-lowering therapy has LDL-Cabove 130 mg/dL. In certain such embodiments the individual in need oflipid-lowering therapy has LDL-C above 100 mg/dL. In certain suchembodiments the individual in need of lipid-lowering therapy shouldmaintain LDL-C below 160 mg/dL. In certain such embodiments theindividual in need of lipid-lowering therapy should maintain LDL-C below130 mg/dL. In certain such embodiments the individual in need oflipid-lowering therapy should maintain LDL-C below 100 mg/dL. In certainsuch embodiments the individual should maintain LDL-C below 70 mg/dL.

In certain embodiments the invention provides methods for reducing ApoBin an individual. In certain embodiments the invention provides methodsfor reducing ApoB-containing lipoprotein in an individual. In certainembodiments the invention provides methods for reducing LDL-C in anindividual. In certain embodiments the invention provides methods forreducing VLDL-C in an individual. In certain embodiments the inventionprovides methods for reducing IDL-C in an individual. In certainembodiments the invention provides methods for reducing non-HDL-C in anindividual. In certain embodiments the invention provides methods forreducing Lp(a) in an individual. In certain embodiments the inventionprovides methods for reducing serum triglyceride in an individual. Incertain embodiments the invention provides methods for reducing livertriglyceride in an individual. In certain embodiments the inventionprovides methods for reducing Ox-LDL-C in an individual. In certainembodiments the invention provides methods for reducing small LDLparticles in an individual. In certain embodiments the inventionprovides methods for reducing small VLDL particles in an individual. Incertain embodiments the invention provides methods for reducingphospholipids in an individual. In certain embodiments the inventionprovides methods for reducing oxidized phospholipids in an individual.

In certain embodiments, the methods provided by the present invention donot lower HDL-C. In certain embodiments, the methods provided by thepresent invention do not result in accumulation of lipids in the liver.

In certain embodiments a pharmaceutical composition comprising a shortantisense compound targeted to a PCSK9 nucleic acid is for use intherapy. In certain embodiments, the therapy is the reduction of LDL-C,ApoB, VLDL-C, IDL-C, non-HDL-C, Lp(a), serum triglyceride, livertriglyceride, Ox-LDL-C, small LDL particles, small VLDL, phospholipids,or oxidized phospholipids in an individual. In certain embodiments, thetherapy is the treatment of hypercholesterolemia, mixed dyslipidemia,atherosclerosis, a risk of developing atherosclerosis, coronary heartdisease, a history of coronary heart disease, early onset coronary heartdisease, one or more risk factors for coronary heart disease, type IIdiabetes, type II diabetes with dyslipidemia, dyslipidemia,hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepaticsteatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liverdisease. In additional embodiments, the therapy is the reduction of CHDrisk. In certain the therapy is prevention of atherosclerosis. Incertain embodiments, the therapy is the prevention of coronary heartdisease.

In certain embodiments a pharmaceutical composition comprising a shortantisense compound targeted to a PCSK9 nucleic acid is used for thepreparation of a medicament for reducing LDL-C, ApoB, VLDL-C, IDL-C,non-HDL-C, Lp(a), serum triglyceride, liver triglyceride, Ox-LDL-C,small LDL particles, small VLDL, phospholipids, or oxidizedphospholipids in an individual. In certain embodiments pharmaceuticalcomposition comprising a short antisense compound targeted to PCKS9 isused for the preparation of a medicament for reducing coronary heartdisease risk. In certain embodiments a short antisense compound targetedto a PCSK9 nucleic acid is used for the preparation of a medicament forthe treatment of hypercholesterolemia, mixed dyslipidemia,atherosclerosis, a risk of developing atherosclerosis, coronary heartdisease, a history of coronary heart disease, early onset coronary heartdisease, one or more risk factors for coronary heart disease, type IIdiabetes, type II diabetes with dyslipidemia, dyslipidemia,hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepaticsteatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liverdisease.

PCSK9 Combination Therapies

In certain embodiments, one or more pharmaceutical compositions of thepresent invention are co-administered with one or more otherpharmaceutical agents. In certain embodiments, such one or more otherpharmaceutical agents are designed to treat the same disease orcondition as the one or more pharmaceutical compositions of the presentinvention. In certain embodiments, such one or more other pharmaceuticalagents are designed to treat a different disease or condition as the oneor more pharmaceutical compositions of the present invention. In certainembodiments, such one or more other pharmaceutical agents are designedto treat an undesired effect of one or more pharmaceutical compositionsof the present invention. In certain embodiments, one or morepharmaceutical compositions of the present invention are co-administeredwith another pharmaceutical agent to treat an undesired effect of thatother pharmaceutical agent. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are administered at the same time. Incertain embodiments, one or more pharmaceutical compositions of thepresent invention one or more other pharmaceutical agents areadministered at different times. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are prepared together in a singleformulation. In certain embodiments, one or more pharmaceuticalcompositions of the present invention and one or more otherpharmaceutical agents are prepared separately.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition of the presentinvention include lipid-lowering agents. In certain such embodiments,pharmaceutical agents that may be co-administered with a pharmaceuticalcomposition of the present invention include, but are not limited toatorvastatin, simvastatin, rosuvastatin, and ezetimibe. In certain suchembodiments, the lipid-lowering agent is administered prior toadministration of a pharmaceutical composition of the present invention.In certain such embodiments, the lipid-lowering agent is administeredfollowing administration of a pharmaceutical composition of the presentinvention. In certain such embodiments the lipid-lowering agent isadministered at the same time as a pharmaceutical composition of thepresent invention. In certain such embodiments the dose of aco-administered lipid-lowering agent is the same as the dose that wouldbe administered if the lipid-lowering agent was administered alone. Incertain such embodiments the dose of a co-administered lipid-loweringagent is lower than the dose that would be administered if thelipid-lowering agent was administered alone. In certain such embodimentsthe dose of a co-administered lipid-lowering agent is greater than thedose that would be administered if the lipid-lowering agent wasadministered alone.

In certain embodiments, a co-administered lipid-lowering agent is aHMG-CoA reductase inhibitor. In certain such embodiments the HMG-CoAreductase inhibitor is a statin. In certain such embodiments the statinis selected from atorvastatin, simvastatin, pravastatin, fluvastatin,and rosuvastatin.

In certain embodiments, a co-administered lipid-lowering agent is acholesterol absorption inhibitor. In certain such embodiments,cholesterol absorption inhibitor is ezetimibe.

In certain embodiments, a co-administered lipid-lowering agent is aco-formulated HMG-CoA reductase inhibitor and cholesterol absorptioninhibitor. In certain such embodiments the co-formulated lipid-loweringagent is ezetimibe/simvastatin.

In certain embodiments, a co-administered lipid-lowering agent is amicrosomal triglyceride transfer protein inhibitor (MTP inhibitor).

In certain embodiments, a co-administered lipid-lowering agent is anoligonucleotide targeted to an ApoB nucleic acid.

In certain embodiments, a co-administered pharmaceutical agent is a bileacid sequestrant. In certain such embodiments, the bile acid sequestrantis selected from cholestyramine, colestipol, and colesevelam.

In certain embodiments, a co-administered pharmaceutical agent is anicotinic acid. In certain such embodiments, the nicotinic acid isselected from immediate release nicotinic acid, extended releasenicotinic acid, and sustained release nicotinic acid.

In certain embodiments, a co-administered pharmaceutical agent is afibric acid. In certain such embodiments, a fibric acid is selected fromgemfibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.

Further examples of pharmaceutical agents that may be co-administeredwith a pharmaceutical composition of the present invention include, butare not limited to, corticosteroids, including but not limited toprednisone; immunoglobulins, including, but not limited to intravenousimmunoglobulin (IVIg); analgesics (e.g., acetaminophen);anti-inflammatory agents, including, but not limited to non-steroidalanti-inflammatory drugs (e.g., ibuprofen, COX-1 inhibitors, and COX-2,inhibitors); salicylates; antibiotics; antivirals; antifungal agents;antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins,sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics;hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin,progestin, somatostan, and thyroid hormones); immunomodulators; musclerelaxants; antihistamines; osteoporosis agents (e.g., biphosphonates,calcitonin, and estrogens); prostaglandins, antineoplastic agents;psychotherapeutic agents; sedatives; poison oak or poison sumacproducts; antibodies; and vaccines.

In certain embodiments, the pharmaceutical compositions of the presentinvention may be administered in conduction with a lipid-loweringtherapy. In certain such embodiments, a lipid-lowering therapy istherapeutic lifestyle change. In certain such embodiments, alipid-lowering therapy is LDL apheresis.

Certain Short Antisense Compounds Targeted to a PCSK9 Nucleic Acid

In certain embodiments, short antisense compounds are targeted to aPCSK9 nucleic acid having the sequence of GENBANK® Accession No.NM_(—)174936.2, incorporated herein as SEQ ID NO: 4. In certain suchembodiments, a short antisense compound targeted to SEQ ID NO: 4 is atleast 90% complementary to SEQ ID NO: 4. In certain such embodiments, ashort antisense compound targeted to SEQ ID NO: 4 is at least 95%complementary to SEQ ID NO: 4. In certain such embodiments, a shortantisense compound targeted to SEQ ID NO: 4 is 100% complementary to SEQID NO: 4. In certain embodiments, a short antisense compound targeted toSEQ ID NO: 4 comprises a nucleotide sequence selected from thenucleotide sequences set forth in Table 6 or Table 7.

The nucleotide sequence set forth in each SEQ ID NO in Tables 6 and 7 isindependent of any modification to a sugar moiety, an internucleosidelinkage, or a nucleobase. As such, short antisense compounds defined bya SEQ ID NO may comprise, independently, one or more modifications to asugar moiety, an internucleoside linkage, or a nucleobase. Shortantisense compounds described by Isis Number (Isis NO.) indicate acombination of nucleobase sequence and one or more modifications to asugar moiety, an internucleoside linkage, or a nucleobase.

Tables 6 and 7 illustrate examples of short antisense compounds targetedto SEQ ID NO: 4. Table 6 illustrates short antisense compounds that are100% complementary to SEQ ID NO: 4. Table 7 illustrates short antisensecompounds that have one or two mismatches with respect to SEQ ID NO: 4.The column labeled ‘gapmer motif’ indicates the wing-gap-wing motif ofeach short antisense compounds. The gap segment comprises2′-deoxynucleotides and each nucleotide of each wing segment comprises a2′-modified sugar. The particular 2′-modified sugar is also indicated inthe ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flankedby wing segments of two nucleotides, where the nucleotides of the wingsegments are 2′-MOE nucleotides. Internucleoside linkages arephosphorothioate. The short antisense compounds comprise5-methylcytidine in place of unmodified cytosine, unless “unmodifiedcytosine” is listed in the gapmer motif column, in which case theindicated cytosines are unmodified cytosines. For example, “5-mC in gaponly” indicates that the gap segment has 5-methylcytosines, while thewing segments have unmodified cytosines.

TABLE 6 Short Antisense Compounds targeted to SEQ ID NO: 4 5′ 3′ SEQISIS Target Target Sequence ID NO. Site Site (5′-3′) Gapmer Motif NO400297 695 708 ATGGGGCAACTTCA 2-10-2 MOE 329 400298 696 709CATGGGGCAACTTC 2-10-2 MOE 330 400299 697 710 ACATGGGGCAACTT 2-10-2 MOE331 400300 742 755 GGGATGCTCTGGGC 2-10-2 MOE 332 400301 757 770CGCTCCAGGTTCCA 2-10-2 MOE 333 400302 828 841 GATACACCTCCACC 2-10-2 MOE334 400303 829 842 AGATACACCTCCAC 2-10-2 MOE 335 400304 830 843GAGATACACCTCCA 2-10-2 MOE 336 400305 937 950 GCCTGTCTGTGGAA 2-10-2 MOE337 400306 952 965 CTGTCACACTTGCT 2-10-2 MOE 338 400307 988 1001CGGCCGCTGACCAC 2-10-2 MOE 339 400308 989 1002 CCGGCCGCTGACCA 2-10-2 MOE340 400309 990 1003 CCCGGCCGCTGACC 2-10-2 MOE 341 400310 991 1004TCCCGGCCGCTGAC 2-10-2 MOE 342 400311 992 1005 ATCCCGGCCGCTGA 2-10-2 MOE343 400312 993 1006 CATCCCGGCCGCTG 2-10-2 MOE 344 400313 994 1007GCATCCCGGCCGCT 2-10-2 MOE 345 400314 1057 1070 GTGCCCTTCCCTTG 2-10-2 MOE346 400315 1075 1088 ATGAGGGTGCCGCT 2-10-2 MOE 347 400316 1076 1089TATGAGGGTGCCGC 2-10-2 MOE 348 400317 1077 1090 CTATGAGGGTGCCG 2-10-2 MOE349 400318 1078 1091 CCTATGAGGGTGCC 2-10-2 MOE 350 400319 1093 1106CGAATAAACTCCAG 2-10-2 MOE 351 400320 1094 1107 CCGAATAAACTCCA 2-10-2 MOE352 400321 1095 1108 TCCGAATAAACTCC 2-10-2 MOE 353 400322 1096 1109TTCCGAATAAACTC 2-10-2 MOE 354 400323 1147 1160 GCCAGGGGCAGCAG 2-10-2 MOE355 400324 1255 1268 GAGTAGAGGCAGGC 2-10-2 MOE 356 400325 1334 1347CCCCAAAGTCCCCA 2-10-2 MOE 357 400326 1335 1348 TCCCCAAAGTCCCC 2-10-2 MOE358 400327 1336 1349 GTCCCCAAAGTCCC 2-10-2 MOE 359 400328 1453 1466ACGTGGGCAGCAGC 2-10-2 MOE 360 400329 1454 1467 CACGTGGGCAGCAG 2-10-2 MOE361 400330 1455 1468 CCACGTGGGCAGCA 2-10-2 MOE 362 400331 1456 1469GCCACGTGGGCAGC 2-10-2 MOE 363 400332 1569 1582 CAGGGAACCAGGCC 2-10-2 MOE364 400333 1570 1583 TCAGGGAACCAGGC 2-10-2 MOE 365 400334 1571 1584CTCAGGGAACCAGG 2-10-2 MOE 366 400335 1572 1585 CCTCAGGGAACCAG 2-10-2 MOE367 400336 1573 1586 TCCTCAGGGAACCA 2-10-2 MOE 368 400337 1574 1587GTCCTCAGGGAACC 2-10-2 MOE 369 400338 1575 1588 GGTCCTCAGGGAAC 2-10-2 MOE370 400339 1576 1589 TGGTCCTCAGGGAA 2-10-2 MOE 371 400340 1577 1590CTGGTCCTCAGGGA 2-10-2 MOE 372 400341 1578 1591 GCTGGTCCTCAGGG 2-10-2 MOE373 400342 1621 1634 GTGCTGGGGGGCAG 2-10-2 MOE 374 400343 1622 1635GGTGCTGGGGGGCA 2-10-2 MOE 375 400344 1623 1636 GGGTGCTGGGGGGC 2-10-2 MOE376 400345 1624 1637 TGGGTGCTGGGGGG 2-10-2 MOE 377 400346 1738 1751GAGCAGCTCAGCAG 2-10-2 MOE 378 400347 1739 1752 GGAGCAGCTCAGCA 2-10-2 MOE379 400348 1740 1753 TGGAGCAGCTCAGC 2-10-2 MOE 380 400349 1741 1754CTGGAGCAGCTCAG 2-10-2 MOE 381 400350 1834 1847 CCCTCACCCCCAAA 2-10-2 MOE382 400351 1835 1848 ACCCTCACCCCCAA 2-10-2 MOE 383 400352 1836 1849CACCCTCACCCCCA 2-10-2 MOE 384 400353 1837 1850 ACACCCTCACCCCC 2-10-2 MOE385 400354 1838 1851 GACACCCTCACCCC 2-10-2 MOE 386 400355 1839 1852AGACACCCTCACCC 2-10-2 MOE 387 400356 1840 1853 TAGACACCCTCACC 2-10-2 MOE388 400357 2083 2096 TGGCAGCAGGAAGC 2-10-2 MOE 389 400358 2084 2097ATGGCAGCAGGAAG 2-10-2 MOE 390 400359 2085 2098 CATGGCAGCAGGAA 2-10-2 MOE391 400360 2086 2099 GCATGGCAGCAGGA 2-10-2 MOE 392 400361 2316 2329GGCAGCAGATGGCA 2-10-2 MOE 393 400362 2317 2330 CGGCAGCAGATGGC 2-10-2 MOE394 400363 2318 2331 CCGGCAGCAGATGG 2-10-2 MOE 395 400364 2319 2332TCCGGCAGCAGATG 2-10-2 MOE 396 400365 2320 2333 CTCCGGCAGCAGAT 2-10-2 MOE397 400366 2321 2334 GCTCCGGCAGCAGA 2-10-2 MOE 398 400367 2322 2335GGCTCCGGCAGCAG 2-10-2 MOE 399 400368 2323 2336 CGGCTCCGGCAGCA 2-10-2 MOE400 400369 2324 2337 CCGGCTCCGGCAGC 2-10-2 MOE 401 400370 2325 2338GCCGGCTCCGGCAG 2-10-2 MOE 402 400371 3543 3556 AGTTACAAAAGCAA 2-10-2 MOE403 403739 988 1001 CGGCCGCTGACCAC 2-10-2 339 (6′S)-6′- methyl-Methyleneoxy BNA 403740 1455 1468 CCACGTGGGCAGCA 2-10-2 362 (6′S)-6′-methyl- Methyleneoxy BNA

TABLE 7 Short antisense compounds targeted to SEQ ID NO: 4 and having 1or 2 mismatches 5′ 3′ SEQ ISIS Target Target Sequence ID NO. Site Site(5′-3′) Gapmer Motif NO 400323 349 362 GCCAGGGGCAGCAG 2-10-2 MOE 355400370 679 692 GCCGGCTCCGGCAG 2-10-2 MOE 402 400361 1860 1873GGCAGCAGATGGCA 2-10-2 MOE 393 400323 1873 1886 GCCAGGGGCAGCAG 2-10-2 MOE355 400310 2257 2270 TCCCGGCCGCTGAC 2-10-2 MOE 342 400361 2653 2666GGCAGCAGATGGCA 2-10-2 MOE 393 400350 2811 2824 CCCTCACCCCCAAA 2-10-2 MOE382 400351 2812 2825 ACCCTCACCCCCAA 2-10-2 MOE 383 400352 2813 2826CACCCTCACCCCCA 2-10-2 MOE 384 400353 2814 2827 ACACCCTCACCCCC 2-10-2 MOE385 400334 2966 2979 CTCAGGGAACCAGG 2-10-2 MOE 366 400332 3379 3392CAGGGAACCAGGCC 2-10-2 MOE 364 400340 3448 3461 CTGGTCCTCAGGGA 2-10-2 MOE372 400341 3449 3462 GCTGGTCCTCAGGG 2-10-2 MOE 373

In certain embodiments, a target region is nucleotides 695-710 of SEQ IDNO: 4. In certain such embodiments, short antisense compounds targetedto nucleotides 695-710 of SEQ ID NO: 4 comprise a nucleotide sequenceselected from SEQ ID NO: 329, 330, or 331. In certain such embodiments,a short antisense compound targeted to nucleotides 695-710 of SEQ ID NO:4 is selected from Isis NO. 400297, 400298, or 400299.

In certain embodiments, a target region is nucleotides 742-770 of SEQ IDNO: 4. In certain such embodiments, a short antisense compound targetedto nucleotides 742-770 of SEQ ID NO: 4 comprises a nucleotide sequenceselected from SEQ ID NO 332 or 333. In certain such embodiments, a shortantisense compound targeted to nucleotides 742-770 of SEQ ID NO: 4 isselected from Isis NO. 400300 or 400301.

In certain embodiments, a target region is nucleotides 828-843 of SEQ IDNO: 4. In certain such embodiments, a short antisense compound targetedto nucleotides 828-843 of SEQ ID NO: 4 comprises a nucleotide sequenceselected from SEQ ID NO 334, 335, or 336. In certain such embodiments, ashort antisense compound targeted to nucleotides 828-843 of SEQ ID NO: 4is selected from ISIS No. 400302, 400303, or 400304.

In certain embodiments, a target region is nucleotides 937-1007 of SEQID NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 937-1007 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 337, 338, 339, 340, 341, 342, 343, 344,or 345. In certain such embodiments, a short antisense compound targetedto nucleotides 937-1007 of SEQ ID NO: 4 is selected from Isis NO.400305, 400306, 400307, 400308, 400309, 400310, 400311, 400312, 400313,or 403739.

In certain embodiments, a target region is nucleotides 937-965 of SEQ IDNO: 4. In certain such embodiments, a short antisense compound targetedto nucleotides 937-965 of SEQ ID NO: 4 comprises a nucleotide sequenceselected from SEQ ID NO 337 or 338. In certain such embodiments, a shortantisense compound targeted to nucleotides 937-965 of SEQ ID NO: 4 isselected from Isis NO. 400305 or 400306.

In certain embodiments, a target region is nucleotides 988-1007 of SEQID NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 988-1007 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 339, 340, 341, 342, 343, 344, or 345.In certain such embodiments, a short antisense compound targeted tonucleotides 937-1007 of SEQ ID NO: 4 is selected from Isis NO. 400307,400308, 400309, 400310, 400311, 400312, 4003313, or 403739.

In certain embodiments, a target region is nucleotides 1057-1160 of SEQID NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1057-1160 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 346, 347, 348, 349, 350, 351, 352, 353,354, or 355. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1057-1160 of SEQ ID NO: 4 is selected from ISISNO: 400314, 400315, 400316, 400317, 400318, 400319, 400320, 400321,400322, or 400323.

In certain embodiments, a target region is nucleotides 1057-1109 of SEQID NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1057-1109 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 346, 347, 348, 349, 350, 351, 352, 353,or 354. In certain such embodiments, a short antisense compound targetedto nucleotides 1057-1109 of SEQ ID NO: 4 is selected from ISIS NO.400314, 400315, 400316, 400317, 400318, 400319, 400320, 400321, or400322.

In certain embodiments, a target region is nucleotides 1057-1091 of SEQID NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1057-1091 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 346, 347, 348, 349, or 350. In certainsuch embodiments, a short antisense compound targeted to nucleotides1057-1091 of SEQ ID NO: 4 is selected from ISIS NO. 400314, 400315,400316, 400317, or 400318.

In certain embodiments, a target region is nucleotides 1093-1109 of SEQID NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1093-1109 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 351, 352, 353, or 354. In certain suchembodiments, a short antisense compound targeted to nucleotides1057-1109 of SEQ ID NO: 4 is selected from ISIS NO: 400319, 400320,400321, or 400322.

In certain embodiments, a target region is nucleotides 1334-1349 of SEQID NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1334-1349 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 357, 358, or 359. In certain suchembodiments, a short antisense compound targeted to nucleotides1334-1349 of SEQ ID NO: 4 is selected from ISIS NO 400325, 400326, or400327.

In certain embodiments, a target region is nucleotides 1453-1469 of SEQID. NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1453-1469 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 360, 361, 362, or 363. In certain suchembodiments, a short antisense compound targeted to nucleotides1453-1469 of SEQ ID NO: 4 is selected from ISIS NO 400328, 400329,400330, 400331, or 403-470.

In certain embodiments, a target region is nucleotides 1569-1591 of SEQID NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1569-1591 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 364, 365, 366, 367, 368, 369, 370, 371,372, or 373. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1569-1591 of SEQ ID NO: 4 is selected from ISISNO 400332, 400333, 400334, 400335, 400336, 400337, 400338, 400339,400340, or 400341.

In certain embodiments, a target region is nucleotides 1621-1637 of SEQID NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1621-1637 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 374, 375, 376, or 377. In certain suchembodiments, a short antisense compound targeted to nucleotides1621-1637 of SEQ ID NO: 4 is selected from ISIS NO 400342, 400343,400344, or 400345.

In certain embodiments, a target region is nucleotides 1738-1754 of SEQID NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1738-1754 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 378, 379, 380, or 381. In certain suchembodiments, a short antisense compound targeted to nucleotides1738-1754 of SEQ ID NO: 4 is selected from ISIS NO 400346, 400347,400348, or 400349.

In certain embodiments, a target region is nucleotides 1834-1853 of SEQID NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1834-1853 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 382, 383, 384, 385, 386, 387, or 388.In certain embodiments, a short antisense compound targeted tonucleotides 1834-1853 of SEQ ID NO: 4 is selected from ISIS NO 400350,400351, 400352, 400353, 400354, 400355, or 400356.

In certain embodiments, a target region is nucleotides 2083-2099 of SEQID NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 2083-2099 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 389, 390, 391, or 392. In certain suchembodiments, a short antisense compound targeted to nucleotides2083-2099 of SEQ ID NO: 4 is selected from ISIS NO 400357, 400358,400359, or 400360.

In certain embodiments, a target region is nucleotides 2316-2338 of SEQID NO: 4. In certain such embodiments, a short antisense compoundtargeted to nucleotides 2316-2338 of SEQ ID NO: 4 comprises a nucleotidesequence selected from SEQ ID NO 393, 394, 395, 396, 397, 398, 399, 400,401, or 402. In certain such embodiments, a short antisense compoundtargeted to nucleotides 2316-2338 of SEQ ID NO: 4 is selected from ISISNO 400361, 400362, 400363, 400364, 400365, 400366, 400367, 400368,400369, or 400370.

In certain embodiments, short antisense compounds targeted to a PCSK9nucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 nucleotides in length. In certain embodiments,short antisense compounds targeted to a PCSK9 nucleic acid are 9 to 14nucleotides in length. In certain embodiments, short antisense compoundstargeted to a PCSK9 nucleic acid are 10 to 14 nucleotides in length. Incertain embodiments, such short antisense compounds are short antisenseoligonucleotides.

In certain embodiments, short antisense compounds targeted to a PCSK9nucleic acid are short gapmers. In certain such embodiments, shortgapmers targeted to a PCSK9 nucleic acid comprise at least one highaffinity modification in one or more wings of the compound. In certainembodiments, short antisense compounds targeted to a PCSK9 nucleic acidcomprise 1 to 3 high-affinity modifications in each wing. In certainsuch embodiments, the nucleosides or nucleotides of the wing comprise a2′ modification. In certain such embodiments, the monomers of the wingare BNA's. In certain such embodiments, the monomers of the wing areselected from α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, β-D-Methyleneoxy(4′-CH₂—O-2′) BNA, Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, Aminooxy(4′-CH₂—O—N(R)-2′) BNA and Oxyamino (4′-CH₂—N(R)—O-2′) BNA. In certainembodiments, the monomers of a wing comprise a substituent at the 2′position selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H or substituted or unsubstitutedC₁-C₁₀ alkyl. In certain embodiments, the monomers of a wing are 2′MOEnucleotides.

In certain embodiments, short antisense compounds targeted to a PCSK9nucleic acid comprise a gap between the 5′ wing and the 3′ wing. Incertain embodiments the gap comprises five, six, seven, eight, nine,ten, eleven, twelve, thirteen, or fourteen monomers. In certainembodiments, the monomers of the gap are unmodifieddeoxyribonucleotides. In certain embodiments, the monomers of the gapare unmodified ribonucleotides. In certain embodiments, gapmodifications (if any) gap result in an antisense compound that, whenbound to its target nucleic acid, supports cleavage by an RNase,including, but not limited to, RNase H.

In certain embodiments, short antisense compounds targeting a PCSK9nucleic acid may have any one or more properties or characteristics ofthe short antisense compounds generally described herein. In certainembodiments, short antisense compounds targeting a PCSK9 nucleic acidhave a motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2,2-10-1-1, 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1,3-6-3 or 1-6-1, more preferably 1-10-1, 2-10-2, 3-10-3, and 1-9-2.

In certain embodiments, short antisense compounds targeted to a PCSK9nucleic acid have uniform monomeric linkages. In certain suchembodiments, those linkages are all phosphorothioate linkages. Incertain embodiments, the linkages are all phosphodiester linkages. Incertain embodiments, short antisense compounds targeted to a PCSK9nucleic acid have mixed backbones.

In certain embodiments, short antisense compounds targeted to a PCSK9nucleic acid are 8 monomers in length. In certain embodiments, shortantisense compounds targeted to a PCSK9 nucleic acid are 9 monomers inlength. In certain embodiments, short antisense compounds targeted to aPCSK9 nucleic acid are 10 monomers in length. In certain embodiments,short antisense compounds targeted to a PCSK9 nucleic acid are 11monomers in length. In certain embodiments, short antisense compoundstargeted to a PCSK9 nucleic acid are monomers in length. In certainembodiments, short antisense compounds targeted to a PCSK9 nucleic acidare 13 monomers in length. In certain embodiments, short antisensecompounds targeted to a PCSK9 nucleic acid are 14 monomers in length. Incertain embodiments, short antisense compounds targeted to a PCSK9nucleic acid are 15 monomers in length. In certain embodiments, shortantisense compounds targeted to a PCSK9 nucleic acid are 16 monomers inlength. In certain embodiments, short antisense compounds targeted to aPCSK9 nucleic acid comprise 9 to 15 monomers. In certain embodiments,short antisense compounds targeted to a PCSK9 nucleic acid comprise 10to 15 monomers. In certain embodiments, short antisense compoundstargeted to a PCSK9 nucleic acid comprise 12 to 14 monomers. In certainembodiments, short antisense compounds targeted to a PCSK9 nucleic acidcomprise 12 to 14 nucleotides or nucleosides.

In certain embodiments, the invention provides methods of modulatingexpression of PCSK9. In certain embodiments, such methods comprise useof one or more short antisense compound targeted to a PCSK9 nucleicacid, wherein the short antisense compound targeted to a PCSK9 nucleicacid is from about 8 to about 16, preferably 9 to 15, more preferably 9to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16linked monomers). One of ordinary skill in the art will appreciate thatthis comprehends methods of modulating expression of PCSK9 using one ormore short antisense compounds targeted to a PCSK9 nucleic acid of 8, 9,10, 11, 12, 13, 14, 15 or 16 monomers.

In certain embodiments, methods of modulating PCSK9 comprise use of ashort antisense compound targeted to a PCSK9 nucleic acid that is 8monomers in length. In certain embodiments, methods of modulating PCSK9comprise use of a short antisense compound targeted to a PCSK9 nucleicacid that is 9 monomers in length. In certain embodiments, methods ofmodulating PCSK9 comprise use of a short antisense compound targeted toa PCSK9 nucleic acid that is 10 monomers in length. In certainembodiments, methods of modulating PCSK9 comprise use of a shortantisense compound targeted to a PCSK9 nucleic acid that is 11 monomersin length. In certain embodiments, methods of modulating PCSK9 compriseuse of a short antisense compound targeted to a PCSK9 nucleic acid thatis 12 monomers in length. In certain embodiments, methods of modulatingPCSK9 comprise use of a short antisense compound targeted to a PCSK9nucleic acid that is 13 monomers in length. In certain embodiments,methods of modulating PCSK9 comprise use of a short antisense compoundtargeted to a PCSK9 nucleic acid that is 14 monomers in length. Incertain embodiments, methods of modulating PCSK9 comprise use of a shortantisense compound targeted to a PCSK9 nucleic acid that is 15 monomersin length. In certain embodiments, methods of modulating PCSK9 compriseuse of a short antisense compound targeted to a PCSK9 nucleic acid thatis 16 monomers in length.

In certain embodiments, methods of modulating expression of PCSK9comprise use of a short antisense compound targeted to a PCSK9 nucleicacid comprising 9 to 15 monomers. In certain embodiments, methods ofmodulating expression of PCSK9 comprise use of a short antisensecompound targeted to a PCSK9 nucleic acid comprising 10 to 15 monomers.In certain embodiments, methods of modulating expression of PCSK9comprise use of a short antisense compound targeted to a PCSK9 nucleicacid comprising 12 to 14 monomers. In certain embodiments, methods ofmodulating expression of PCSK9 comprise use of a short antisensecompound targeted to a PCSK9 nucleic acid comprising 12 or 14nucleotides or nucleosides.

4. Superoxide Dismutase 1 Enzyme (SOD1)

The enzymes known as the superoxide dismutases (SODs) provide defenseagainst oxidative damage of biomolecules by catalyzing the dismutationof superoxide to hydrogen peroxide (H₂O₂) (Fridovich, Annu. Rev.Biochem., 1995, 64, 97-112). Two major classes of superoxide dismutasesexist. One consists of a group of enzymes with active sites containingcopper and zinc while the other class has either manganese or iron atthe active site (Fridovich, Annu. Rev. Biochem., 1995, 64, 97-112).

Mutations in the superoxide dismutase 1 gene are associated with adominantly-inherited form of amyotrophic lateral sclerosis (ALS, alsoknown as Lou Gehrig's disease) a disorder characterized by a selectivedegeneration of upper and lower motor neurons (Cleveland and Liu, Nat.Med., 2000, 6, 1320-1321). The deleterious effects of various mutationson superoxide dismutase 1 are most likely mediated through a gain oftoxic function rather than a loss of superoxide dismutase 1 activity, asthe complete absence of superoxide dismutase 1 in mice neitherdiminishes life nor provokes overt disease (Al-Chalabi and Leigh, Curr.Opin. Neurol., 2000, 13, 397405; Alisky and Davidson, Hum. Gene Ther.,2000, 11, 2315-2329).

Over 100 mutations of the human SOD1 gene have been identified, andaltogether account for approximately 20% of familial amyotrophic lateralsclerosis (ALS) cases. Some mutations, such as the A4V mutation mostcommonly found in the United States, are highly lethal and result insurvival only nine months from the onset of disease symptoms. Othermutations of SOD1 manifest in a slower disease course.

Definitions

“SOD1” means the gene product or protein of which expression is to bemodulated by administration of a short antisense compound.

“SOD1 nucleic acid” means any nucleic acid encoding SOD1. For example,in certain embodiments, a SOD1 nucleic acid includes, withoutlimitations, a DNA sequence encoding SOD1, an RNA sequence transcribedfrom DNA encoding SOD1, and an mRNA sequence encoding SOD1.

“SOD1 mRNA” means an mRNA encoding SOD1.

SOD1 Therapeutic Indications

It has been discovered that antisense inhibition of superoxide dismutase1 (SOD1) in an animal model of familial ALS reduces both SOD1 mRNA andprotein, and further results in a slowing of disease progression and,importantly, increased survival time. Accordingly, in certainembodiments, the invention provides methods for the slowing of diseaseprogression in an individual suffering from familial ALS byadministering to such an individual a short antisense compound targetedto an SOD1 nucleic acid. In certain such embodiments, a short antisensecompound targeted to SOD1 are delivered directly to the cerebrospinalfluid of the individual. In certain such embodiments, methods furthercomprise increasing survival time of an individual suffering fromfamilial ALS. Slowing of disease progression is indicated by animprovement in one or more indicators of ALS disease progression,including, without limitation, the revised ALS functional rating scale,pulmonary function tests, and muscle strength measurements.

SOD1 Combination Therapies

In certain embodiments, one or more pharmaceutical compositionscomprising a short antisense compound targeted to an SOD1 nucleic acidis co-administered with one or more other pharmaceutical agents. Incertain embodiments, such one or more other pharmaceutical agents aredesigned to treat the same disease or condition as the one or morepharmaceutical compositions of the present invention. In certainembodiments, such one or more other pharmaceutical agents are designedto treat a different disease or condition as the one or morepharmaceutical compositions of the present invention. In certainembodiments, such one or more other pharmaceutical agents are designedto treat an undesired effect of one or more pharmaceutical compositionsof the present invention. In certain embodiments, one or morepharmaceutical compositions of the present invention are co-administeredwith another pharmaceutical agent to treat an undesired effect of thatother pharmaceutical agent. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are administered at the same time. Incertain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areadministered at different times. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are prepared together in a singleformulation. In certain embodiments, one or more pharmaceuticalcompositions of the present invention and one or more otherpharmaceutical agents are prepared separately.

In certain embodiments, a co-administered pharmaceutical agent is anicotinic acid. In certain such embodiments, the nicotinic acid isselected from immediate release nicotinic acid, extended releasenicotinic acid, and sustained release nicotinic acid.

In certain embodiments, a co-administered pharmaceutical agent is afibric acid. In certain such embodiments, a fibric acid is selected fromgemfibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.

Further examples of pharmaceutical agents that may be co-administeredwith a pharmaceutical composition comprising a short antisense compoundtargeted to SOD1 include, but are not limited to, corticosteroids,including but not limited to prednisone; immunoglobulins, including, butnot limited to intravenous immunoglobulin (IVIg); analgesics (e.g.,acetaminophen); anti-inflammatory agents, including, but not limited tonon-steroidal anti-inflammatory drugs (e.g., ibuprofen, COX-1inhibitors, and COX-2, inhibitors); salicylates; antibiotics;antivirals; antifungal agents; antidiabetic agents (e.g., biguanides,glucosidase inhibitors, insulins, sulfonylureas, andthiazolidenediones); adrenergic modifiers; diuretics; hormones (e.g.,anabolic steroids, androgen, estrogen, calcitonin, progestin,somatostan, and thyroid hormones); immunomodulators; muscle relaxants;antihistamines; osteoporosis agents (e.g., biphosphonates, calcitonin,and estrogens); prostaglandins, antineoplastic agents; psychotherapeuticagents; sedatives; poison oak or poison sumac products; antibodies; andvaccines.

Certain Short Antisense Compounds Targeted to a SOD1 Nucleic Acid

In certain embodiments, short antisense compounds are targeted to a SOD1nucleic acid having the sequence of GENBANK® Accession No.NM_(—×02317.1), incorporated herein as SEQ ID NO: 5. In certain suchembodiments, a short antisense compound targeted to SEQ ID NO: 5 is atleast 90% complementary to SEQ ID NO: 5. In certain such embodiments, ashort antisense compound targeted to SEQ ID NO: 5 is at least 95%complementary to SEQ ID NO: 5. In certain such embodiments, a shortantisense compound targeted to SEQ ID NO: 5 is 100% complementary to SEQID NO: 5. In certain embodiments, a short antisense compound targeted toSEQ ID NO: 5 comprises a nucleotide sequence selected from thenucleotide sequences set forth in Table 8 or Table 9.

The nucleotide sequence set forth in each SEQ ID NO in Tables 8 and 9 isindependent of any modification to a sugar moiety, an internucleosidelinkage, or a nucleobase. As such, short antisense compounds defined bya SEQ ID NO may comprise, independently, one or more modifications to asugar moiety, an internucleoside linkage, or a nucleobase. Shortantisense compounds described by Isis Number (Isis NO.) indicate acombination of nucleobase sequence and one or more modifications to asugar moiety, an internucleoside linkage, or a nucleobase.

Table 8 illustrates examples of short antisense compounds targeted toSEQ ID NO: 5. Table 8 illustrates short antisense compounds that are100% complementary to SEQ ID NO: 5. The column labeled ‘gapmer motif’indicates the wing-gap-wing motif of each short antisense compounds. Thegap segment comprises 2′-deoxynucleotides and each nucleotide of eachwing segment comprises a 2′-modified sugar. The particular 2′-modifiedsugar is also indicated in the ‘gapmer motif’ column. For example,‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten2′-deoxynucleotides is flanked by wing segments of two nucleotides,where the nucleotides of the wing segments are 2′-MOE nucleotides.Internucleoside linkages are phosphorothioate. The short antisensecompounds comprise 5-methylcytidine in place of unmodified cytosine,unless “unmodified cytosine” is listed in the gapmer motif column, inwhich case the indicated cytosines are unmodified cytosines. Forexample, “5-mC in gap only” indicates that the gap segment has5-methylcytosines, while the wing segments have unmodified cytosines.

In certain embodiments, short antisense compounds targeting a SOD1nucleic acid may have any one or more properties or characteristics ofthe short antisense compounds generally described herein. In certainembodiments, short antisense compounds targeting a SOD1 nucleic acidhave a motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2,2-10-1-1, 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1,3-6-3 or 1-6-1, more preferably 1-10-1, 2-10-2, 3-10-3, and 1-9-2.

TABLE 8 Short Antisense Compounds targeted to SEQ ID NO: 5 5′ 3′ SEQISIS Target Target Gapmer ID NO. Site Site Sequence (5′-3′) Motif NO387541 85 100 GTCGCCCTTCAGCACG 3-10-3 MOE 406 387540 86 99TCGCCCTTCAGCAC 2-10-2 MOE 407 387539 87 98 CGCCCTTCAGCA 1-10-1 MOE 408

In certain embodiments, a target region is nucleotides 85-100 of SEQ IDNO: 5. In certain such embodiments, short antisense compounds targetedto nucleotides 85-100 of SEQ ID NO: 5 comprise a nucleotide sequenceselected from SEQ ID NO: 406, 407, or 408. In certain such embodiments,a short antisense compound targeted to nucleotides 85-100 of SEQ ID NO:5 is selected from Isis No. 387541, 387540, or 387539.

In certain embodiments, short antisense compounds targeted to a SOD1nucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 nucleotides in length. In certain embodiments,short antisense compounds targeted to a SOD1 nucleic acid are 9 to 14nucleotides in length. In certain embodiments, short antisense compoundstargeted to a SOD1 nucleic acid are 10 to 14 nucleotides in length. Incertain embodiments, such short antisense compounds are short antisenseoligonucleotides.

In certain embodiments, short antisense compounds targeted to a SOD1nucleic acid are short gapmers. In certain such embodiments, shortgapmers targeted to a SOD1 nucleic acid comprise at least one highaffinity modification in one or more wings of the compound. In certainembodiments, short antisense compounds targeted to a SOD1 nucleic acidcomprise 1 to 3 high-affinity modifications in each wing. In certainsuch embodiments, the nucleosides or nucleotides of the wing comprise a2′ modification. In certain such embodiments, the monomers of the wingare BNA's. In certain such embodiments, the monomers of the wing areselected from α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, β-D-Methyleneoxy(4′-CH₂—O-2′) BNA, Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, Aminooxy(4′-CH₂—O—N(R)-2′) BNA and Oxyamino (4′-CH₂—N(R)—O-2′) BNA. In certainembodiments, the monomers of a wing comprise a substituent at the 2′position selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H or substituted or unsubstitutedC₁-C₁₀ alkyl. In certain embodiments, the monomers of a wing are 2′MOEnucleotides.

In certain embodiments, short antisense compounds targeted to a SOD1nucleic acid comprise a gap between the 5′ wing and the 3′ wing. Incertain embodiments the gap comprises five, six, seven, eight, nine,ten, eleven, twelve, thirteen, or fourteen monomers. In certainembodiments, the monomers of the gap are unmodifieddeoxyribonucleotides. In certain embodiments, the monomers of the gapare unmodified ribonucleotides. In certain embodiments, gapmodifications (if any) gap result in an antisense compound that, whenbound to its target nucleic acid, supports cleavage by an RNase,including, but not limited to, RNase H.

In certain embodiments, short antisense compounds targeted to a SOD1nucleic acid have uniform monomeric linkages. In certain suchembodiments, those linkages are all phosphorothioate linkages. Incertain embodiments, the linkages are all phosphodiester linkages. Incertain embodiments, short antisense compounds targeted to a SOD1nucleic acid have mixed backbones.

In certain embodiments, short antisense compounds targeted to a SOD1nucleic acid are 8 monomers in length. In certain embodiments, shortantisense compounds targeted to a SOD1 nucleic acid are 9 monomers inlength. In certain embodiments, short antisense compounds targeted to aSOD1 nucleic acid are 10 monomers in length. In certain embodiments,short antisense compounds targeted to a SOD1 nucleic acid are 11monomers in length. In certain embodiments, short antisense compoundstargeted to a SOD1 nucleic acid are monomers in length. In certainembodiments, short antisense compounds targeted to a SOD1 nucleic acidare 13 monomers in length. In certain embodiments, short antisensecompounds targeted to a SOD1 nucleic acid are 14 monomers in length. Incertain embodiments, short antisense compounds targeted to a SOD1nucleic acid are 15 monomers in length. In certain embodiments, shortantisense compounds targeted to a SOD1 nucleic acid are 16 monomers inlength. In certain embodiments, short antisense compounds targeted to aSOD1 nucleic acid comprise 9 to 15 monomers. In certain embodiments,short antisense compounds targeted to a SOD1 nucleic acid comprise 10 to15 monomers. In certain embodiments, short antisense compounds targetedto a SOD1 nucleic acid comprise 12 to 14 monomers. In certainembodiments, short antisense compounds targeted to a SOD1 nucleic acidcomprise 12 to 14 nucleotides or nucleosides.

In certain embodiments, the invention provides methods of modulatingexpression of SOD1. In certain embodiments, such methods comprise use ofone or more short antisense compound targeted to a SOD1 nucleic acid,wherein the short antisense compound targeted to a SOD1 nucleic acid isfrom about 8 to about 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linkedmonomers). One of ordinary skill in the art will appreciate that thiscomprehends methods of modulating expression of SOD1 using one or moreshort antisense compounds targeted to a SOD1 nucleic acid of 8, 9, 10,11, 12, 13, 14, 15 or 16 monomers.

In certain embodiments, methods of modulating SOD1 comprise use of ashort antisense compound targeted to a SOD1 nucleic acid that is 8monomers in length. In certain embodiments, methods of modulating SOD1comprise use of a short antisense compound targeted to a SOD1 nucleicacid that is 9 monomers in length. In certain embodiments, methods ofmodulating SOD1 comprise use of a short antisense compound targeted to aSOD1 nucleic acid that is 10 monomers in length. In certain embodiments,methods of modulating SOD1 comprise use of a short antisense compoundtargeted to a SOD1 nucleic acid that is 11 monomers in length. Incertain embodiments, methods of modulating SOD1 comprise use of a shortantisense compound targeted to a SOD1 nucleic acid that is 12 monomersin length. In certain embodiments, methods of modulating SOD1 compriseuse of a short antisense compound targeted to a SOD1 nucleic acid thatis 13 monomers in length. In certain embodiments, methods of modulatingSOD1 comprise use of a short antisense compound targeted to a SOD1nucleic acid that is 14 monomers in length. In certain embodiments,methods of modulating SOD1 comprise use of a short antisense compoundtargeted to a SOD1 nucleic acid that is 15 monomers in length. Incertain embodiments, methods of modulating SOD1 comprise use of a shortantisense compound targeted to a SOD1 nucleic acid that is 16 monomersin length.

In certain embodiments, methods of modulating expression of SOD1comprise use of a short antisense compound targeted to a SOD1 nucleicacid comprising 9 to 15 monomers. In certain embodiments, methods ofmodulating expression of SOD1 comprise use of a short antisense compoundtargeted to a SOD1 nucleic acid comprising 10 to 15 monomers. In certainembodiments, methods of modulating expression of SOD1 comprise use of ashort antisense compound targeted to a SOD1 nucleic acid comprising 12to 14 monomers. In certain embodiments, methods of modulating expressionof SOD1 comprise use of a short antisense compound targeted to a SOD1nucleic acid comprising 12 or 14 nucleotides or nucleosides.

5. CRP

CRP (also known as C-reactive protein and PTX1) is an essential humanacute-phase reactant produced in the liver in response to a variety ofinflammatory cytokines. The protein, first identified in 1930, is highlyconserved and considered to be an early indicator of infectious orinflammatory conditions. Plasma CRP levels increase 1.000-fold inresponse to infection, ischemia, trauma, burns, and inflammatoryconditions. In clinical trials where patients receive lipid-loweringtherapy, such as statin therapy, it has been demonstrated that patientshaving reductions in both LDL-C and CRP have a reduced risk of futurecoronary events relative to patients experiencing only reductions inLDL-C.

Definitions

“CRP” means the gene product or protein of which expression is to bemodulated by a short antisense compound.

“CRP nucleic acid” means any nucleic acid encoding CRP. For example, incertain embodiments, a CRP nucleic acid includes, without limitations, aDNA sequence encoding CRP, an RNA sequence transcribed from DNA encodingCRP, and an mRNA sequence encoding CRP.

“CRP mRNA” means an mRNA encoding CRP.

CRP Therapeutic Indications

In certain embodiments, the invention provides methods of modulating CRPexpression in an individual comprising administering to the individual ashort antisense compound targeted to a CRP nucleic acid. In certainembodiments, the invention provides methods of treating an individualcomprising administering one or more pharmaceutical compositionscomprising a short antisense compound targeted to a CRP nucleic acid. Incertain embodiments, the individual has hypercholesterolemia,non-familial hypercholesterolemia, familial hypercholesterolemia,heterozygous familial hypercholesterolemia, homozygous familialhypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk ofdeveloping atherosclerosis, coronary heart disease, a history ofcoronary heart disease, early onset coronary heart disease, one or morerisk factors for coronary heart disease. In certain embodiments, theindividual has acute coronary syndrome, vascular injury, arterialocclusion, unstable angina, post peripheral vascular disease, postmyocardial infarction (MI), thrombosis, deep vein thrombus, end-stagerenal disease (ESRD), chronic renal failure, complement activation,congestive heart failure, or systemic vasculitis. In certainembodiments, the individual has had a stroke.

In certain embodiments, the individual has undergone a procedureselected from elective stent placement, angioplasty, post percutaneoustransluminal angioplasty (PTCA), cardiac transplantation, renal dialysisor cardiopuhnonary bypass.

In certain embodiments, the individual has an inflammatory disease. Incertain such embodiments, the inflammatory disease is selected frominflammatory bowel disease, ulcerative colitis, rheumatoid arthritis, orosteoarthritis.

Guidelines for lipid-lowering therapy were established in 2001 by AdultTreatment Panel III (ATP III) of the National Cholesterol EducationProgram (NCEP), and updated in 2004 (Grundy et al., Circulation, 2004,110, 227-239). The guidelines include obtaining a complete lipoproteinprofile, typically after a 9 to 12 hour fast, for determination ofLDL-C, total cholesterol, and HDL-C levels. According to the mostrecently established guidelines, LDL-C levels of 130-159 mg/dL, 160-189mg/dL, and greater than or equal to 190 mg/dL are considered borderlinehigh, high, and very high, respectively. Total cholesterol levels of200-239 and greater than or equal to 240 mg/dL are considered borderlinehigh and high, respectively. HDL-C levels of less than 40 mg/dL areconsidered low.

In certain embodiments, the individual has been identified as in need oflipid-lowering therapy. In certain such embodiments, the individual hasbeen identified as in need of lipid-lowering therapy according to theguidelines established in 2001 by Adult Treatment Panel III (ATP 111) ofthe National Cholesterol Education Program (NCEP), and updated in 2004(Grundy et al., Circulation, 2004, 110, 227-239). In certain suchembodiments, the individual in need of lipid-lowering therapy has LDL-Cabove 190 mg/dL. In certain such embodiments, the individual in need oflipid-lowering therapy has LDL-C above 160 mg/dL. In certain suchembodiments, the individual in need of lipid-lowering therapy has LDL-Cabove 130 mg/dL. In certain such embodiments the individual in need oflipid-lowering therapy has LDL-C above 100 mg/dL. In certain suchembodiments the individual in need of lipid-lowering therapy shouldmaintain LDL-C below 160 mg/dL. In certain such embodiments theindividual in need of lipid-lowering therapy should maintain LDL-C below130 mg/dL. In certain such embodiments the individual in need oflipid-lowering therapy should maintain LDL-C below 100 mg/dL. In certainsuch embodiments the individual should maintain LDL-C below 70 mg/dL.

In certain embodiments the invention provides methods for reducing CRPin an individual. In certain such embodiments, the reduction in CRP isat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, and at least 100%.

In certain embodiments, the methods provided by the present invention donot lower HDL-C. In certain embodiments, the methods provided by thepresent invention do not result in accumulation of lipids in the liver.In certain embodiments, the methods provided by the present invention donot cause hepatic steatosis.

In certain embodiments, the invention provides methods for lowering CRPconcentration in a subject while reducing side effects associated withtreatment. In certain such embodiments, a side effect is liver toxicity.In certain such embodiments, a side effect is abnormal liver function.In certain such embodiments, a side effect is elevated alanineaminotransferase (ALT). In certain such embodiments, a side effect iselevated aspartate aminotransferase (AST).

In certain embodiments, the invention provides methods for lowering CRPconcentration in a subject who is not reaching target LDL-C levels as aresult of lipid-lowering therapy. In certain such embodiments, a shortantisense compound targeted to a CRP nucleic acid is the onlypharmaceutical agent administered to the subject. In certain suchembodiments, the subject has not complied with recommendedlipid-lowering therapy. In certain such embodiments, a pharmaceuticalcomposition of the invention is co-administered with an additionaldifferent lipid-lowering therapy. In certain such embodiments, anadditional lipid-lowering therapy is LDL-apheresis. In certain suchembodiments, an additional lipid-lowering therapy is a statin. Incertain such embodiments, an additional lipid-lowering therapy isezetimibe.

In certain embodiments, the invention provides methods for lowering CRPconcentration in a statin-intolerant subject. In certain suchembodiments, the subject has creatine kinase concentration increases asa result of statin administration. In certain such embodiments, thesubject has liver function abnormalities as a result of statinadministration. In certain such embodiments the subject has muscle achesas a result of statin administration. In certain such embodiments thesubject has central nervous system side effects as a result of statinadministration. In certain embodiments, the subject has not compliedwith recommended statin administration.

In certain embodiments, the invention provides methods for reducingcoronary heart disease risk in a subject. In certain embodiments theinvention provides methods for slowing the progression ofatherosclerosis in a subject. In certain such embodiments the inventionprovides methods for stopping the progression of atherosclerosis in asubject. In certain such embodiments the invention provides methods forreducing the size and/or prevalence of atherosclerotic plaques in asubject. In certain embodiments the methods provided reduce a subject'srisk of developing atherosclerosis.

In certain embodiments the methods provided improve the cardiovascularoutcome in a subject. In certain such embodiments improvedcardiovascular outcome is the reduction of the risk of developingcoronary heart disease. In certain such embodiments, improvedcardiovascular outcome is a reduction in the occurance of one or moremajor cardiovascular events, which include, but are not limited to,death, myocardial infarction, reinfarction, stroke, cardiogenic shock,pulmonary edema, cardiac arrest, and atrial dysrhythmia. In certain suchembodiments, the improved cardiovascular outcome is evidenced byimproved carotid intimal media thickness. In certain such embodiments,improved carotid intimal media thickness is a decrease in thickness. Incertain such embodiments, improved carotid intimal media thickness is aprevention an increase of intimal media thickness.

In certain embodiments a pharmaceutical composition comprising a shortantisense compound targeted to a CRP nucleic acid is for use in therapy.In certain embodiments, the therapy is the reduction of CRP in anindividual. In certain embodiments, the therapy is the treatment ofhypercholesterolemia, non-familial hypercholesterolemia, familialhypercholesterolemia, heterozygous familial hypercholesterolemia,homozygous familial hypercholesterolemia, mixed dyslipidemia,atherosclerosis, a risk of developing atherosclerosis, coronary heartdisease, a history of coronary heart disease, or early onset coronaryheart disease. In additional embodiments, the therapy is the reductionof CHD risk. In certain the therapy is prevention of atherosclerosis. Incertain embodiments, the therapy is the prevention of coronary heartdisease. In certain embodiments, the therapy is the treatment of acutecoronary syndrome, chronic renal failure, vascular injury, arterialocclusion, atherothrombosis, unstable angina, post peripheral vasculardisease, post myocardial infarction (MI), thrombosis, deep veinthrombus, end-stage renal disease (ESRD), complement activation,congestive heart failure, or systemic vasculitis. In certain embodimentsthe therapy is the treatment of an individual who has undergone aprocedure selected from elective stent placement, angioplasty, postpercutaneous transluminal angioplasty (PTCA), cardiac transplantation,renal dialysis or cardiopulmonary bypass. In certain embodiments, thetherapy is the treatment of an inflammatory disorder.

In certain embodiments a pharmaceutical composition comprising a shortantisense compound targeted to a CRP nucleic acid is used for thepreparation of a medicament for reducing CRP in an individual. Incertain embodiments pharmaceutical composition comprising a shortantisense compound targeted to a CRP nucleic acid is used for thepreparation of a medicament for reducing coronary heart disease risk. Incertain embodiments a short antisense compound targeted to a CRP nucleicacid is used for the preparation of a medicament for the treatment ofhypercholesterolemia, non-familial hypercholesterolemia, familialhypercholesterolemia, heterozygous familial hypercholesterolemia,homozygous familial hypercholesterolemia, mixed dyslipidemia,atherosclerosis, a risk of developing atherosclerosis, coronary heartdisease, a history of coronary heart disease, early onset coronary heartdisease, or one or more risk factors for coronary heart disease.

In certain embodiments, a short antisense compound targeted to a CRPnucleic acid is used for the preparation of a medicament for thetreatment of acute coronary syndrome, chronic renal failure, vascularinjury, arterial occlusion, atherothrombosis, unstable angina, postperipheral vascular disease, post myocardial infarction (MI),thrombosis, deep vein thrombus, end-stage renal disease (ESRD),complement activation, congestive heart failure, or systemic vasculitis.In certain embodiments, a short antisense compound targeted to a CRPnucleic acid is used for the preparation of a medicament for thetreatment of an individual who has had a stroke.

In certain embodiments, a short antisense compound targeted to a CRPnucleic acid is used for the preparation of a medicament for thetreatment in an individual who has undergone a procedure selected fromelective stent placement, angioplasty, post percutaneous transluminalangioplasty (PTCA), cardiac transplantation, renal dialysis orcardiopulmonary bypass.

In certain embodiments, a short antisense compound targeted to a CRPnucleic acid is used for the preparation of a medicament for thetreatment of an inflammatory disease. In certain such embodiments, ashort antisense compound targeted to a CRP nucleic acid is used for thepreparation of a medicament for the treatment of inflammatory boweldisease, ulcerative colitis, rheumatoid arthritis, or osteoarthritis.

CRP Combination Therapies

In certain embodiments, one or more pharmaceutical compositionscomprising a short antisense compound targeted to a CRP nucleic acid areco-administered with one or more other pharmaceutical agents. In certainembodiments, the one or more other pharmaceutical agents is alipid-lowering agent. In certain embodiments, such one or more otherpharmaceutical agents are designed to treat the same disease conditionas the one or more pharmaceutical compositions of the present invention.In certain embodiments, such one or more other pharmaceutical agents aredesigned to treat a different disease or condition as the one or morepharmaceutical compositions of the present invention. In certainembodiments, such one or more other pharmaceutical agents are designedto treat an undesired effect of one or more pharmaceutical compositionsof the present invention. In certain embodiments, one or morepharmaceutical compositions of the present invention are co-administeredwith another pharmaceutical agent to treat an undesired effect of thatother pharmaceutical agent. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are administered at the same time. Incertain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areadministered at different times. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are prepared together in a singleformulation. In certain embodiments, one or more pharmaceuticalcompositions of the present invention and one or more otherpharmaceutical agents are prepared separately.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition comprising a shortantisense compound targeted to a CRP nucleic acid include lipid-loweringagents. In certain such embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition of the presentinvention include, but are not limited to atorvastatin, simvastatin,rosuvastatin, and ezetimibe. In certain such embodiments, thelipid-lowering agent is administered prior to administration of apharmaceutical composition of the present invention. In certain suchembodiments, the lipid-lowering agent is administered followingadministration of a pharmaceutical composition of the present invention.In certain such embodiments the lipid-lowering agent is administered atthe same time as a pharmaceutical composition of the present invention.In certain such embodiments the dose of a co-administered lipid-loweringagent is the same as the dose that would be administered if thelipid-lowering agent was administered alone. In certain such embodimentsthe dose of a co-administered lipid-lowering agent is lower than thedose that would be administered if the lipid-lowering agent wasadministered alone. In certain such embodiments the dose of aco-administered lipid-lowering agent is greater than the dose that wouldbe administered if the lipid-lowering agent was administered alone.

In certain embodiments, a co-administered lipid-lowering agent is aHMG-CoA reductase inhibitor. In certain such embodiments the HMG-CoAreductase inhibitor is a statin. In certain such embodiments the statinis selected from atorvastatin, simvastatin, pravastatin, fluvastatin,and rosuvastatin.

In certain embodiments, a co-administered lipid-lowering agent is ISIS301012.

In certain embodiments, a co-administered lipid-lowering agent is acholesterol absorption inhibitor. In certain such embodiments,cholesterol absorption inhibitor is ezetimibe.

In certain embodiments, a co-administered lipid-lowering agent is aco-formulated HMG-CoA reductase inhibitor and cholesterol absorptioninhibitor. In certain such embodiments the co-formulated lipid-loweringagent is ezetimibe/simvastatin.

In certain embodiments, a co-administered lipid-lowering agent is amicrosomal triglyceride transfer protein inhibitor (MTP inhibitor).

In certain embodiments, a co-administered pharmaceutical agent is a bileacid sequestrant. In certain such embodiments, the bile acid sequestrantis selected from cholestyramine, colestipol, and colesevelam.

In certain embodiments, a co-administered pharmaceutical agent is anicotinic acid. In certain such embodiments, the nicotinic acid isselected from immediate release nicotinic acid, extended releasenicotinic acid, and sustained release nicotinic acid.

In certain embodiments, a co-administered pharmaceutical agent is afibric acid. In certain such embodiments, a fibric acid is selected fromgemfibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.

Further examples of pharmaceutical agents that may be co-administeredwith a pharmaceutical composition comprising a short antisense compoundtargeted to a CRP nucleic acid include, but are not limited to,corticosteroids, including but not limited to prednisone;immunoglobulins, including, but not limited to intravenousimmunoglobulin (IVIg); analgesics (e.g., acetaminophen);anti-inflammatory agents, including, but not limited to non-steroidalanti-inflammatory drugs (e.g., ibuprofen, COX-1 inhibitors, and COX-2,inhibitors); salicylates; antibiotics; antivirals; antifungal agents;antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins,sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics;hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin,progestin, somatostan, and thyroid hormones); immunomodulators; musclerelaxants; antihistamines; osteoporosis agents (e.g., biphosphonates,calcitonin, and estrogens); prostaglandins, antineoplastic agents;psychotherapeutic agents; sedatives; poison oak or poison sumacproducts; antibodies; and vaccines.

In certain embodiments, a pharmaceutical composition comprising a shortantisense compound targeted to a CRP nucleic acid may be administered inconjuction with a lipid-lowering therapy. In certain such embodiments, alipid-lowering therapy is therapeutic lifestyle change. In certain suchembodiments, a lipid-lowering therapy is LDL apheresis.

Certain Short Antisense Compounds Targeted to a CRP Nucleic Acid

In certain embodiments, short antisense compounds are targeted to a CRPnucleic acid having the sequence of GENBANK® Accession No.NM_(—)000567.1, incorporated herein as SEQ ID NO: 6. In certain suchembodiments, a short antisense compound targeted to SEQ ID NO: 6 is atleast 90% complementary to SEQ ID NO: 6. In certain such embodiments, ashort antisense compound targeted to SEQ ID NO: 6 is at least 95%complementary to SEQ ID NO: 6. In certain such embodiments, a shortantisense compound targeted to SEQ ID NO: 6 is 100% complementary to SEQID NO: 6. In certain embodiments, a short antisense compound targeted toSEQ ID NO: 6 comprises a nucleotide sequence selected from thenucleotide sequences set forth in Table 9.

The nucleotide sequence set forth in each SEQ ID NO in Table 9 isindependent of any modification to a sugar moiety, an internucleosidelinkage, or a nucleobase. As such, short antisense compounds defined bya SEQ ID NO may comprise, independently, one or more modifications to asugar moiety, an internucleoside linkage, or a nucleobase. Shortantisense compounds described by Isis Number (Isis NO.) indicate acombination of nucleobase sequence and one or more modifications to asugar moiety, an internucleoside linkage, or a nucleobase.

Table 9 illustrates examples of short antisense compounds targeted toSEQ ID NO: 6. Table 9 illustrates short antisense compounds that are100% complementary to SEQ ID NO: 6. The column labeled ‘gapmer motif’indicates the wing-gap-wing motif of each short antisense compounds. Thegap segment comprises 2′-deoxynucleotides and each nucleotide of eachwing segment comprises a 2′-modified sugar. The particular 2′-modifiedsugar is also indicated in the ‘gapmer motif’ column. For example,‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten2′-deoxynucleotides is flanked by wing segments of two nucleotides,where the nucleotides of the wing segments are 2′-MOE nucleotides.Internucleoside linkages are phosphorothioate. The short antisensecompounds comprise 5-methylcytidine in place of unmodified cytosine,unless “unmodified cytosine” is listed in the gapmer motif column, inwhich case the indicated cytosines are unmodified cytosines. Forexample, “5-mC in gap only” indicates that the gap segment has5-methylcytosines, while the wing segments have unmodified cytosines.

In certain embodiments, short antisense compounds targeting a CRPnucleic acid may have any one or more properties or characteristics ofthe short antisense compounds generally described herein. In certainembodiments, short antisense compounds targeting a CRP nucleic acid havea motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2, 2-10-1-1,3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or1-6-1, more preferably 1-10-1, 2-10-2, 3-10-3, and 1-9-2.

TABLE 9 Short Antisense Compounds targeted to SEQ ID NO: 6 5′ 3′ SeqISIS Target Target Gapmer ID NO. Site Site Sequence (5′-3′) Motif NO353506 1257 1272 ACTCTGGACCCAAACC 3-10-3 MOE 409 353507 1258 1271CTCTGGACCCAAAC 2-10-2 MOE 410 353484 1305 1320 CCATTTCAGGAGACCT 3-10-3MOE 411 353485 1306 1319 CATTTCAGGAGACC 2-10-2 MOE 412

In certain embodiments, a target region is nucleotides 1305-1320 ofNM_(—)000567.1. In certain such embodiments, short antisense compoundstargeted to nucleotides 1305-1320 of NM_(—)000567.1 comprise anucleotide sequence selected from SEQ ID NO: 1305 or 1306. In certainsuch embodiments, a short antisense compound targeted to nucleotides263-278 of NM-000567.1 is selected from Isis NO. 353484 or 353485.

In certain embodiments, a target region is nucleotides 1257-1272 ofNM-000567.1. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1257-1272 of NM_(—)000567.1 comprises anucleotide sequence selected from SEQ ID NO 1257 or 1258. In certainsuch embodiments, a short antisense compound targeted to nucleotides428-483 of NM-000567.1 is selected from Isis NO. 353506 or 353507.

In certain embodiments, short antisense compounds targeted to a CRPnucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 nucleotides in length. In certain embodiments,short antisense compounds targeted to a CRP nucleic acid are 9 to 14nucleotides in length. In certain embodiments, short antisense compoundstargeted to a CRP nucleic acid are 10 to 14 nucleotides in length. Incertain embodiments, such short antisense compounds are short antisenseoligonucleotides.

In certain embodiments, short antisense compounds targeted to a CRPnucleic acid are short gapmers. In certain such embodiments, shortgapmers targeted to a CRP nucleic acid comprise at least one highaffinity modification in one or more wings of the compound. In certainembodiments, short antisense compounds targeted to a CRP nucleic acidcomprise 1 to 3 high-affinity modifications in each wing. In certainsuch embodiments, the nucleosides or nucleotides of the wing comprise a2′ modification. In certain such embodiments, the monomers of the wingare BNA's. In certain such embodiments, the monomers of the wing areselected from α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, β-D-Methyleneoxy(4′-CH₂—O-2′) BNA, Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, Aminooxy(4′-CH₂—O—N(R)-2′) BNA and Oxyamino (4′-CH₂—N(R)—O-2′) BNA. In certainembodiments, the monomers of a wing comprise a substituent at the 2′position selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H or substituted or unsubstitutedC₁-C₁₀ alkyl. In certain embodiments, the monomers of a wing are 2′MOEnucleotides.

In certain embodiments, short antisense compounds targeted to a CRPnucleic acid comprise a gap between the 5′ wing and the 3′ wing. Incertain embodiments the gap comprises five, six, seven, eight, nine,ten, eleven, twelve, thirteen, or fourteen monomers. In certainembodiments, the monomers of the gap are unmodifieddeoxyribonucleotides. In certain embodiments, the monomers of the gapare unmodified ribonucleotides. In certain embodiments, gapmodifications (if any) gap result in an antisense compound that, whenbound to its target nucleic acid, supports cleavage by an RNase,including, but not limited to, RNase H.

In certain embodiments, short antisense compounds targeted to a CRPnucleic acid have uniform monomeric linkages. In certain suchembodiments, those linkages are all phosphorothioate linkages. Incertain embodiments, the linkages are all phosphodiester linkages. Incertain embodiments, short antisense compounds targeted to a CRP nucleicacid have mixed backbones.

In certain embodiments, short antisense compounds targeted to a CRPnucleic acid are 8 monomers in length. In certain embodiments, shortantisense compounds targeted to a CRP nucleic acid are 9 monomers inlength. In certain embodiments, short antisense compounds targeted to aCRP nucleic acid are 10 monomers in length. In certain embodiments,short antisense compounds targeted to a CRP nucleic acid are 11 monomersin length. In certain embodiments, short antisense compounds targeted toa CRP nucleic acid are monomers in length. In certain embodiments, shortantisense compounds targeted to a CRP nucleic acid are 13 monomers inlength. In certain embodiments, short antisense compounds targeted to aCRP nucleic acid are 14 monomers in length. In certain embodiments,short antisense compounds targeted to a CRP nucleic acid are 15 monomersin length. In certain embodiments, short antisense compounds targeted toa CRP nucleic acid are 16 monomers in length. In certain embodiments,short antisense compounds targeted to a CRP nucleic acid comprise 9 to15 monomers. In certain embodiments, short antisense compounds targetedto a CRP nucleic acid comprise 10 to 15 monomers. In certainembodiments, short antisense compounds targeted to a CRP nucleic acidcomprise 12 to 14 monomers. In certain embodiments, short antisensecompounds targeted to a CRP nucleic acid comprise 12 to 14 nucleotidesor nucleosides.

In certain embodiments, the invention provides methods of modulatingexpression of CRP. In certain embodiments, such methods comprise use ofone or more short antisense compound targeted to a CRP nucleic acid,wherein the short antisense compound targeted to a CRP nucleic acid isfrom about 8 to about 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linkedmonomers). One of ordinary skill in the art will appreciate that thiscomprehends methods of modulating expression of CRP using one or moreshort antisense compounds targeted to a CRP nucleic acid of 8, 9, 10,11, 12, 13, 14, 15 or 16 monomers.

In certain embodiments, methods of modulating CRP comprise use of ashort antisense compound targeted to a CRP nucleic acid that is 8monomers in length. In certain embodiments, methods of modulating CRPcomprise use of a short antisense compound targeted to a CRP nucleicacid that is 9 monomers in length. In certain embodiments, methods ofmodulating CRP comprise use of a short antisense compound targeted to aCRP nucleic acid that is 10 monomers in length. In certain embodiments,methods of modulating CRP comprise use of a short antisense compoundtargeted to a CRP nucleic acid that is 11 monomers in length. In certainembodiments, methods of modulating CRP comprise use of a short antisensecompound targeted to a CRP nucleic acid that is 12 monomers in length.In certain embodiments, methods of modulating CRP comprise use of ashort antisense compound targeted to a CRP nucleic acid that is 13monomers in length. In certain embodiments, methods of modulating CRPcomprise use of a short antisense compound targeted to a CRP nucleicacid that is 14 monomers in length. In certain embodiments, methods ofmodulating CRP comprise use of a short antisense compound targeted to aCRP nucleic acid that is 15 monomers in length. In certain embodiments,methods of modulating CRP comprise use of a short antisense compoundtargeted to a CRP nucleic acid that is 16 monomers in length.

In certain embodiments, methods of modulating expression of CRP compriseuse of a short antisense compound targeted to a CRP nucleic acidcomprising 9 to 15 monomers. In certain embodiments, methods ofmodulating expression of CRP comprise use of a short antisense compoundtargeted to a CRP nucleic acid comprising 10 to 15 monomers. In certainembodiments, methods of modulating expression of CRP comprise use of ashort antisense compound targeted to a CRP nucleic acid comprising 12 to14 monomers. In certain embodiments, methods of modulating expression ofCRP comprise use of a short antisense compound targeted to a CRP nucleicacid comprising 12 or 14 nucleotides or nucleosides.

6. Glucocorticoid Receptor (GCCR)

Glucocorticoids were among the first steroid hormones to be identifiedand are responsible for a multitude of physiological functions,including the stimulation of gluconeogenesis, decreased glucose uptakeand utilization in peripheral tissues, increased glycogen deposition,suppression of immune and inflammatory responses, inhibition of cytokinesynthesis and acceleration of various developmental events.Glucocorticoids are also especially important for combating stress.Stress-induced elevation of glucocorticoid synthesis and release leadsto, among other responses, increased ventricular workload, inhibition ofinflammatory mediators, inhibition of cytokine synthesis and increasedglucose production (Karin, Cell, 1998, 93, 487-490).

Both natural glucocorticoids and their synthetic derivatives exert theiraction through the glucocorticoid receptor, a ubiquitously expressedcytoplasmic member of the nuclear hormone superfamily of receptors.Human glucocorticoid receptor is also known as nuclear receptorsubfamily 3, group C, member 1; NR3C1; GCCR; GCR; GRL; Glucocorticoidreceptor, lymphocyte. The gene is located on human chromosome 5q11-q13and consists of 9 exons (Encio and Detera-Wadleigh, J Biol Chem, 1991,266, 7182-7188; Gehring et al., Proc Natl Acad Sci USA, 1985, 82,3751-3755). Multiple forms of human glucocorticoid receptor mRNA exist:a 5.5 kb human glucocorticoid receptor α cDNA containing exons 1-8 andexon 9α; a 4.3 kb human glucocorticoid receptor β cDNA containing exons1-8 and exon 9β; and a 7.0 kb human glucocorticoid receptor a cDNAcontaining exons 1-8 and the entire exon 9, which includes exon 9α, exon90 and the ‘J region’, which is flanked by exons 9α and 9β (Hollenberget al., Nature, 1985, 318, 635-641; Oakley et al., J Biol Chem, 1996,271, 9550-9559). Human glucocorticoid receptor a is the predominantisoform of the receptor and the one that exhibits steroid bindingactivity (Hollenberg et al., Nature, 1985, 318, 635-641). Additionally,through usage of three different promoters three different exon 1variants can be transcribed, and alternative splicing of one exon 1variant can result in three different versions of this exon. Thus, humanglucocorticoid receptor mRNA may contain 5 different versions of exon 1(Breslin et al., Mol Endocrinol, 2001, 15, 1381-1395).

Examination of the expression patterns of the α and β isoforms of humanglucocorticoid receptor mRNA reveals that the a isoform is moreabundantly expressed. Both isoforms are expressed in similar tissues andcell types, including lung, kidney, heart, liver, skeletal muscle,macrophages, neutrophils and peripheral blood mononuclear cells. Onlyhuman glucocorticoid receptor a is expressed in colon. At the level ofprotein, while the a isoform is detected in all tissues examined, the 13isoform is undetectable, suggesting that under physiological conditions,the default splicing pathway is the one that produces the a isoform(Pujols et al., Am J Physiol Cell Physiol, 2002, 283, C1324-1331). The βisoform of glucocorticoid receptor binds neither a glucocorticoidagonist nor an antagonist. Furthermore, the β isoform is localizedprimarily in the nucleus in transfected cells, independent of hormonestimulation. When both isoforms are expressed in the same cell, theglucocorticoid receptor P inhibits the hormone-induced, glucocorticoidreceptor α-mediated stimulation of gene expression, suggesting that theβ isoform functions as an inhibitor of glucocorticoid receptor aactivity (Oakley et al., J Biol Chem, 1996, 271, 9550-9559). Unlessotherwise noted, the human glucocorticoid receptor described herein isdefined as the ubiquitous product(s) of the gene located on chromosome5q11-q13.

Cell lines transfected with a complementary glucocorticoid receptorantisense RNA strand exhibited a reduction in glucocorticoid receptormRNA levels and a decreased response to the glucocorticoid receptoragonist dexamethasone (Pepin and Barden, Mol Cell Biol, 1991, 11,1647-1653). Transgenic mice bearing an antisense glucocorticoid receptorgene construct were used to study the glucocorticoid feedback effect onthe hypothalamus-pituitary-adrenal axis (Pepin et al., Nature, 1992,355, 725-728). In another study of similarly genetically engineeredmice, energy intake and expenditure, heart and vastus lateralis musclelipoprotein lipase activity, and heart and brown adipose tissuenorepinephrine were lower than in control animals. Conversely, fatcontent and total body energy were significantly higher than in controlanimals. These results suggest that a defective glucocorticoid receptorsystem may affect energy balance through increasing energeticefficiency, and they emphasize the modulatory effects ofhypothalamic-pituitary-adrenal axis changes on muscle lipoprotein lipaseactivity (Richard et al., Am J Physiol, 1993, 265, R146-150).

Behavorial effects of glucocorticoid receptor antagonists have beenmeasured in animal models designed to assess anxiety, learning andmemory. Reduced expression of glucocorticoid receptor in rats long-termintracerebroventricularly infused with antisense oligodeoxynucleotidestargeting glucocorticoid receptor mRNA did not interfere with spatialnavigation in the Morris water maze test (Engelmann et al., Eur JPharmacol, 1998, 361, 17-26). Bilateral infusion of an antisenseoligodeoxynucleotide targeting the glucocorticoid receptor mRNA into thedentate gyrus of the rat hippocampus reduced the immobility of rats inthe Porsolt forced swim test (Korte et al., Eur J Pharmacol, 1996, 301,19-25).

Glucocorticoids are frequently used for their immunosuppressive,anti-inflammatory effects in the treatment of diseases such asallergies, athsma, rheumatoid arthritis, AIDS, systemic lupuserythematosus and degenerative osteoarthritis. Negative regulation ofgene expression, such as that caused by the interaction ofglucocorticoid receptor with NF-kB, is proposed to be at least partlyresponsible for the anti-inflammatory action of glucocorticoids in vivo.Interleukin-6, tumor necrosis factor α and interleukin-1 are the threecytokines that account for most of the hypothalamic-pituitary-adrenal(HPA) axis stimulation during the stress of inflammation. The HPA axisand the systemic sympathetic and adrenomedullary system are theperipheral components of the stress system, responsible for maintainingbasal and stress-related homeostasis. Glucocorticoids, the end productsof the HPA axis, inhibit the production of all three inflammatorycytokines and also inhibit their effects on target tissues, with theexception of interleukin-6, which acts synergistically withglucocorticoids to stimulate the production of acute-phase reactants.Glucocorticoid treatment decreases the activity of the HPA axis(Chrousos, N Engl J Med, 1995, 332, 1351-1362).

In some cases, patients are refractory to glucocorticoid treatment. Onereason for this resistance to steroids lies with mutations orpolymorphisms present in the glucocorticoid receptor gene. A total of 15missense, three nonsense, three frameshift, one splice site, and twoalternative spliced mutations, as well as 16 polymorphisms, have beenreported in the NR3C1 gene in association with glucocorticoid resistance(Bray and Cotton, Hum Mutat, 2003, 21, 557-568). Additional studies inhumans have suggested a positive association between metabolic syndromeincidence and progression, with alleles at the glucocorticoid receptor(GR) gene (Rosmond, Obes Res, 2002, 10, 1078-1086).

Other cases of glucocorticoid insensitivity are associated with alteredexpression of glucocorticoid receptor isoforms. A study of humanglucocorticoid receptor β isoform mRNA expression inglucocorticoid-resistant ulcerative colitis patients revealed thepresence of this mRNA was significantly higher than in theglucocorticoid-sensitive patients, suggesting that the expression ofhuman glucocorticoid receptor β mRNA in the peripheral blood mononuclearcells may serve as a predictor of glucocorticoid response in ulcerativecolitis (Honda et al., Gastroenterology, 2000, 118, 859-866). Increasedexpression of glucocorticoid receptor β is also observed in asignificantly high number of glucocorticoid-insensitive asthmatics.Additionally, cytokine-induced abnormalities in the DNA binding capacityof the glucocorticoid receptor were found in peripheral bloodmononuclear cells from glucocorticoid-insensitive patients transfection,and HepG2 cells with the glucocorticoid receptor P gene resulted in asignificant reduction of glucocorticoid receptor α DNA-binding capacity(Leung et al., J Exp Med, 1997, 186, 1567-1574). Dexamethasone bindingstudies demonstrate that human glucocorticoid receptor P does not alterthe affinity of glucocorticoid receptor α for hormonal ligands, butrather its ability to bind to the GRE (Bamberger et al., J Clin Invest,1995, 95, 2435-2441). Taken together, these results illustrate thatglucocorticoid receptor β, through competition with glucocorticoidreceptor a for GRE target sites, may function as a physiologically andpathophysiologically relevant endogenous inhibitor of glucocorticoidaction.

In the liver, glucocorticoid agonists increase hepatic glucoseproduction by activating the glucocorticoid receptor, which subsequentlyleads to increased expression of the gluconeogenic enzymesphosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase.Through gluconeogenesis, glucose is formed through non-hexoseprecursors, such as lactate, pyruvate and alanine (Link, Curr OpinInvestig Drugs, 2003, 4, 421-429). Steroidal glucocorticoid receptorantagonists such as RU 486 have been tested in rodent models ofdiabetes. Mice deficient in the leptin receptor gene, termed db/db mice,are genetically obese, diabetic and hyperinsulinemic. Treatment ofhyperglycemic db/db mice with RU 486 decreased blood glucose levels byapproximately 49%, without affecting plasma insulin levels.Additionally, RU 486 treatment reduced the expression of glucocorticoidreceptor responsive genes PEPCK, glucose-6-phosphatase, glucosetransporter type 2 and tyrosine aminotransferase in db/db mice ascompared to untreated animals (Friedman et al., J Biol Chem, 1997, 272,31475-31481). RU 486 also ameliorates diabetes in the ob/ob mouse modelof diabetes, obesity and hyperinsulinemia, through a reduction in seruminsulin and blood glucose levels (Gettys et al., Int J Obes Relat MetabDisord, 1997, 21, 865-873).

As increased gluconeogenesis is considered to be the major source ofincreased glucose production in diabetes, a number of therapeutictargets for the inhibition of hepatic glucose production have beeninvestigated. Due to the ability of antagonists of the glucocorticoidreceptor to ameliorate diabetes in animal models, such compounds areamong the potential therapies being explored. However, there aredetrimental systemic effects of glucocorticoid receptor antagonists,including activation of the HPA axis (Link, Curr Opin Investig Drugs,2003, 4, 421-429). Increased HPA axis activity is associated withsuppression of immune-related inflammatory action, which can increasesusceptibility to infectious agents and neoplasms. Conditions associatedwith suppression of immune-mediated inflammation through defects in theHPA axis, or its target tissues, include Cushing's syndrome, chronicstress, chronic alcoholism and melancholic depression (Chrousos, N EnglJ Med, 1995, 332, 1351-1362). Thus, it is of great value to developliver-specific glucocorticoid receptor antagonists. Steroidalglucocorticoid receptor antagonists have been conjugated to bile acidsfor the purpose of targeting them to the liver (Apelqvist et al., 2000).Currently, there are no known therapeutic agents that target theglucocorticoid receptor without undesired peripheral effects (Link, CurrOpin Investig Drugs, 2003, 4, 421-429). Consequently, there remains along felt need for agents capable of effectively inhibiting hepaticglucocorticoid receptor.

Definitions

“Glucocorticoid receptor” is the gene product or protein of whichexpression is to be modulated by administration of a short antisensecompound. Glucocorticoid receptor is generally referred to as GCCR.

“GCCR nucleic acid” means any nucleic acid encoding GCCR. For example,in certain embodiments, a GCCR nucleic acid includes, withoutlimitation, a DNA sequence encoding GCCR, an RNA sequence transcribedfrom DNA encoding GCCR, and an mRNA sequence encoding GCCR.

“GCCR mRNA” means an mRNA encoding GCCR.

Therapeutic Indications

Antisense technology is an effective means of reducing the expression ofspecific gene products and therefore is useful in a number oftherapeutic, diagnostic and research applications for the modulation ofglucocorticoid receptor expression. Furthermore, in certain embodiments,liver is one of the tissues in which the highest concentrations ofantisense oligonucleotides are found following administration (Geary etal., Curr. Opin. Investig. Drugs, 2001, 2, 562-573). Therefore, in suchembodiments, antisense technology represents an attractive method forthe liver-specific inhibition of glucocorticoid receptor.

In certain embodiments, short antisense compounds targeted to a nucleicacid encoding glucocorticoid receptor are preferentially distributed tothe liver. In certain embodiments, short antisense compounds haveincreased potency in the liver when compared to a longer parentcompound. In certain embodiments, target RNA is predominantly expressedin the liver.

For therapeutics, a subject, suspected of having a disease or disorderwhich can be treated by modulating the expression of GCCR is treated byadministering one or more short antisense compound. In a non-limitingexample, the methods comprise the step of administering to an animal atherapeutically effective amount of a short antisense compound. Certainshort antisense compounds inhibit the activity of GCCR and/or inhibitexpression of GCCR. In certain embodiments, the activity or expressionof GCCR in a subject is inhibited by at least 10%, by at least 20%, byat least 25%, by at least 30%, by at least 40%, by at least 50%, by atleast 60%, by at least 70%, by at least 75%, by at least 80%, by atleast 85%, by at least 90%, by at least 95%, by at least 98%, by atleast 99%, or by 100%. In certain embodiments, the activity orexpression of GCCR in a subject is inhibited by at least 30%. In certainembodiments, the activity or expression of GCCR in a subject isinhibited by at least 50% or more.

The reduction of the expression of GCCR may be measured, for example, inblood, plasma, serum, adipose tissue, liver or any other body fluid,tissue or organ of the animal. In certain embodiments, cells containedwithin such fluids, tissues or organs being analyzed comprise nucleicacids encoding GCCR and/or they contain the GCCR protein itself.

Certain pharmaceutical and other compositions comprising short antisensecompounds are also provided. In certain embodiments, short antisensecompounds are be utilized in pharmaceutical compositions by adding tothem an effective amount of a compound to a suitable pharmaceuticallyacceptable diluent or carrier.

In certain embodiments, short antisense compounds targeting a GCCRnucleic acid have any one or more properties or characteristics of theshort antisense compounds generally described herein. In certainembodiments, short antisense compounds targeting a GCCR nucleic acidhave a motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2,2-10-1-1, 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1,3-6-3 or 1-6-1. In certain embodiments, short antisense compoundstargeting a GCCR nucleic acid have a motif (wing-deoxy gap-wing)selected from 1-10-1, 2-10-2, 3-10-3, and 1-9-2. In certain embodiments,short antisense compounds targeting a GCCR nucleic acid have a motif(wing-deoxy gap-wing) selected from 3-10-3, 2-10-3, 2-10-2, 1-10-1,1-10-2, 2-8-2, 1-8-1, 3-6-3 or 1-6-1, more preferably 2-10-2 and 2-8-2.

In certain embodiments, provided herein are methods of treating anindividual by administering one or more short antisense compoundtargeted to a GCCR nucleic acid or a pharmaceutical compositioncomprising such compound. Further provided are methods of treating asubject having a disease or conditions associated with GCCR activity byadministering a short antisense compound targeted to a GCCR nucleicacid. In addition to diabetes, particularly type 2 diabetes, diseasesand conditions associated with GCCR include but are not limited to,obesity, Metabolic syndrome X, Cushing's Syndrome, Addison's disease,inflammatory diseases such as asthma, rhinitis and arthritis, allergy,autoimmune disease, immunodeficiency, anorexia, cachexia, bone loss orbone frailty, and wound healing. Metabolic syndrome, metabolic syndromeX or simply Syndrome X refers to a cluster of risk factors that includeobesity, dyslipidemia, particularly high blood triglycerides, glucoseintolerance, high blood sugar and high blood pressure. In certainembodiments, short antisense compounds targeted to GCCR are used foramelioration of hyperglycemia induced by systemic steroid therapy.Moreover, antisense technology provides a means of inhibiting theexpression of the glucocorticoid receptor P isoform, demonstrated to beoverexpressed in patients refractory to glucocorticoid treatment.

In certain embodiments, the invention provides short antisense compoundstargeted to a nucleic acid encoding GCGR, and which modulate theexpression of glucocorticoid receptor. Pharmaceutical and othercompositions comprising the compounds of the invention are alsoprovided. Further provided are methods of screening for modulators ofglucocorticoid receptor and methods of modulating the expression ofglucocorticoid receptor in cells, tissues or animals comprisingcontacting said cells, tissues or animals with one or more of thecompounds or compositions of the invention. Methods of treating ananimal, particularly a human, suspected of having or being prone to adisease or condition associated with expression of glucocorticoidreceptor are also set forth herein. Such methods comprise administeringa therapeutically or prophylactically effective amount of one or more ofthe compounds or compositions of the invention to the person in need oftreatment.

Certain Short Antisense Compounds Targeted to a GCCR Nucleic Acid.

In certain embodiments, short antisense compounds are targeted to a GCCRnucleic acid having the sequence of nucleotides 1 to 106000 of GENBANK®Accession No. AC012634, incorporated herein as SEQ ID NO: 8. In certainsuch embodiments, a short antisense compound targeted to SEQ ID NO: 8 isat least 90% complementary to SEQ ID NO: 8. In certain such embodiments,a short antisense compound targeted to SEQ ID NO: 8 is at least 95%complementary to SEQ ID NO: 8. In certain such embodiments, a shortantisense compound targeted to SEQ ID NO: 8 is 100% complementary to SEQID NO: 8. In certain embodiments, a short antisense compound targeted toSEQ ID NO: 8 includes a nucleotide sequence selected from the nucleotidesequences set forth in Tables 10 and 11.

The nucleotide sequence set forth in each SEQ ID NO in Tables 10 and 11is independent of any modification to a sugar moiety, an internucleosidelinkage, or a nucleobase. As such, short antisense compounds defined bya SEQ ID NO may comprise, independently, one or more modifications to asugar moiety, an internucleoside linkage, or a nucleobase. Shortantisense compounds described by Isis Number (Isis NO.) indicate acombination of nucleobase sequence and one or more modifications to asugar moiety, an internucleoside linkage, or a nucleobase.

In certain embodiments, short antisense compounds targeted to a GCCRnucleic acid comprise a gapmer motif. In certain embodiments, a shortantisense compound targeted to a GCCR nucleic acid comprises a 2-10-2gapmer motif.

Tables 10 and 11 illustrate examples of short antisense compoundstargeted to SEQ ID NO: 8. Table 10 illustrates short antisense compoundsthat are 100% complementary to SEQ ID NO: 8. Table 11 illustrates shortantisense compounds that have one or two mismatches with respect to SEQID NO: 8. The column labeled ‘gapmer motif’ indicates the wing-gap-wingmotif of each short antisense compounds. The gap segment comprises2′-deoxynucleotides and each nucleotide of each wing segment comprises a2′-modified sugar. The particular 2′-modified sugar is also indicated inthe ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flankedby wing segments of two nucleotides, where the nucleotides of the wingsegments are 2′-MOE nucleotides. Internucleoside linkages arephosphorothioate. The short antisense compounds comprise5-methylcytidine in place of unmodified cytosine, unless “unmodifiedcytosine” is listed in the gapmer motif column, in which case theindicated cytosines are unmodified cytosines. For example, “5-mC in gaponly” indicates that the gap segment has 5-methylcytosines, while thewing segments have unmodified cytosines.

TABLE 10 Short Antisense Compounds targeted to SEQ ID NO: 8 5′ 3′ SEQISIS Target Target Sequence ID NO. Site Site (5′-3′) Gapmer Motif NO371644 88142 88155 TTTGGGAGGTGGTC 2-10-2 MOE 413 371645 88156 88169CACACCAGGCAGAG 2-10-2 MOE 414 371649 88212 88225 CTTTACAGCTTCCA 2-10-2MOE 415 371651 88242 88255 CACTACCTTCCACT 2-10-2 MOE 416 371652 8824888261 AACACACACTACCT 2-10-2 MOE 417 371653 88256 88269 CTCTTCAAAACACA2-10-2 MOE 418 371665 92037 92050 GTAATTGTGCTGTC 2-10-2 MOE 419 37166992086 92099 TTTTTCTTCGAATT 2-10-2 MOE 420 371671 92114 92127CATTTTCGATAGCG 2-10-2 MOE 421 371673 92142 92155 ACCTTCCAGGTTCA 2-10-2MOE 422

TABLE 11 Short antisense compounds targeted to SEQ ID NO: 8 and having 1or 2 mismatches 5′ 3′ SEQ ISIS Target Target Gapmer ID NO Site SiteSequence (5′-3′) Motif NO 371638 2039 2052 ATAGGAAGCATAAA 2-10-2 MOE 423371650 4949 4962 TCTTTTAAAGAAGA 2-10-2 MOE 424 371673 10187 10200ACCTTCCAGGTTCA 2-10-2 MOE 422 371660 13465 13478 AAGGATATTTTAAA 2-10-2MOE 425 371660 14428 14441 AAGGATATTTTAAA 2-10-2 MOE 425 371654 1548615499 GAACAAAAATTAAA 2-10-2 MOE 427 371661 16638 16651 TTCCACAGATCTGT2-10-2 MOE 428 371653 17892 17905 CTCTTCAAAACACA 2-10-2 MOE 418 37167918444 18457 TTTATAAAGTAAAG 2-10-2 MOE 429 371645 19816 19829CACACCAGGCAGAG 2-10-2 MOE 414 371638 21555 21568 ATAGGAAGCATAAA 2-10-2MOE 423 371650 21775 21788 TCTTTTAAAGAAGA 2-10-2 MOE 424 371679 2190221915 TTTATAAAGTAAAG 2-10-2 MOE 429 371655 22507 22520 TACTGTGAGAAATA2-10-2 MOE 433 371655 22722 22735 TACTGTGAGAAATA 2-10-2 MOE 433 37167225662 25675 TTCCAGCTTGAAGA 2-10-2 MOE 435 371678 25926 25939GATCAGTTCTCATG 2-10-2 MOE 436 371655 26041 26054 TACTGTGAGAAATA 2-10-2MOE 433 371638 29770 29783 ATAGGAAGCATAAA 2-10-2 MOE 423 371668 3055130564 TTATCAATGATGCA 2-10-2 MOE 439 371670 40584 40597 GCATGCTGGACAGT2-10-2 MOE 440 371654 43331 43344 GAACAAAAATTAAA 2-10-2 MOE 427 37165046024 46037 TCTTTTAAAGAAGA 2-10-2 MOE 424 371659 50372 50385TTGCACCTGAACTA 2-10-2 MOE 443 371634 50565 50578 CAGAATATATTTCT 2-10-2MOE 444 371673 56942 56955 ACCTTCCAGGTTCA 2-10-2 MOE 422 371654 6237262385 GAACAAAAATTAAA 2-10-2 MOE 427 371679 63537 63550 TTTATAAAGTAAAG2-10-2 MOE 429 371654 64908 64921 GAACAAAAATTAAA 2-10-2 MOE 427 37166165795 65808 TTCCACAGATCTGT 2-10-2 MOE 428 371645 70997 71010CACACCAGGCAGAG 2-10-2 MOE 414 371661 77400 77413 TTCCACAGATCTGT 2-10-2MOE 428 371663 82329 82342 ATAAGAGATTAAAA 2-10-2 MOE 450 371633 8342683439 TCCCCCTTCTCATT 2-10-2 MOE 451 371662 85873 85886 GGGCATTGTTAAAA2-10-2 MOE 452 371654 86476 86489 GAACAAAAATTAAA 2-10-2 MOE 427 37167986516 86529 TTTATAAAGTAAAG 2-10-2 MOE 429 371641 88097 88110AGAACTCACATCTG 2-10-2 MOE 455 371642 88111 88124 GAGCTGGACGGAGG 2-10-2MOE 456 371646 88170 88183 AAGCTTCATCGGAG 2-10-2 MOE 457 371647 8818488197 ATAATGGCATCCCG 2-10-2 MOE 458 371650 88226 88239 TCTTTTAAAGAAGA2-10-2 MOE 424 371673 91493 91506 ACCTTCCAGGTTCA 2-10-2 MOE 422 37166492030 92043 TGCTGTCCTATAAG 2-10-2 MOE 460 371666 92044 92057CACAAAGGTAATTG 2-10-2 MOE 461 371667 92058 92071 ATCATTTCTTCCAG 2-10-2MOE 462 371668 92072 92085 TTATCAATGATGCA 2-10-2 MOE 463 371670 9210092113 GCATGCTGGACAGT 2-10-2 MOE 440 371672 92128 92141 TTCCAGCTTGAAGA2-10-2 MOE 435 371674 92147 92160 CCATTACCTTCCAG 2-10-2 MOE 466 37163792983 92996 GCATAAACAGGGTT 2-10-2 MOE 467 371654 93928 93941GAACAAAAATTAAA 2-10-2 MOE 427 371641 99772 99785 AGAACTCACATCTG 2-10-2MOE 455 371679 99883 99896 TTTATAAAGTAAAG 2-10-2 MOE 429 371660 9993399946 AAGGATATTTTAAA 2-10-2 MOE 425 371635 105004 105017 TATGAAAGGAATGT2-10-2 MOE 472 371654 105028 105041 GAACAAAAATTAAA 2-10-2 MOE 427 371676106482 106495 TTCCTTAAGCTTCC 2-10-2 MOE 474 371650 107838 107851TCTTTTAAAGAAGA 2-10-2 MOE 424 371673 110922 110935 ACCTTCCAGGTTCA 2-10-2MOE 422 371673 111580 111593 ACCTTCCAGGTTCA 2-10-2 MOE 422 371634 114608114621 CAGAATATATTTCT 2-10-2 MOE 444 371638 115040 115053 ATAGGAAGCATAAA2-10-2 MOE 423 371660 116244 116257 AAGGATATTTTAAA 2-10-2 MOE 425 371663116657 116670 ATAAGAGATTAAAA 2-10-2 MOE 450 371673 118068 118081ACCTTCCAGGTTCA 2-10-2 MOE 422 371666 118834 118847 CACAAAGGTAATTG 2-10-2MOE 461 371660 119858 119871 AAGGATATTTTAAA 2-10-2 MOE 425 371660 120210120223 AAGGATATTTTAAA 2-10-2 MOE 425 371662 120876 120889 GGGCATTGTTAAAA2-10-2 MOE 452 371655 124004 124017 TACTGTGAGAAATA 2-10-2 MOE 433 371656124170 124183 GAACAGTTAAACAT 2-10-2 MOE 485

In certain embodiments, a target region is nucleotides 88142-88269 ofSEQ ID NO: 8. In certain embodiments, a short antisense compound istargeted to nucleotides 88142-88269 of SEQ ID NO: 8. In certain suchembodiments, a short antisense compound targeted to nucleotides88142-88269 comprises a nucleotide sequence selected from SEQ ID NO 413,414, 415, 416, 417, or 418. In certain such embodiments, an antisensecompound targeted to nucleotides 88142-88269 of SEQ ID NO: 8 is selectedfrom Isis NO 371644, 371645, 371649, 371651, 371652, or 371653.

In certain embodiments, a target region is nucleotides 88142-88169 ofSEQ ID NO: 8. In certain embodiments, a short antisense compound istargeted to nucleotides 88142-88169 of SEQ ID NO: 8. In certain suchembodiments, a short antisense compound targeted to nucleotides88142-88169 comprises a nucleotide sequence selected from SEQ ID NO 413or 414. In certain such embodiments, an antisense compound targeted tonucleotides 88142-88169 of SEQ ID NO: 8 is selected from Isis NO. 371644or 371645.

In certain embodiments, a target region is nucleotides 88242-88269 ofSEQ ID NO: 8. In certain embodiments, a short antisense compound istargeted to nucleotides 88242-88269 of SEQ ID NO: 8. In certain suchembodiments, a short antisense compound targeted to nucleotides88242-88269 comprises a nucleotide sequence selected from SEQ ID NO 416,417, or 418. In certain such embodiments, an antisense compound targetedto nucleotides 88242-88269 of SEQ ID NO: 8 is selected from Isis NO.371651, 371652, or 371653.

In certain embodiments, a target region is nucleotides 92037-92155 ofSEQ ID NO: 8. In certain embodiments, a short antisense compound istargeted to nucleotides 92037-92155 of SEQ ID NO: 8. In certain suchembodiments, a short antisense compound targeted to nucleotides92037-92155 comprises a nucleotide sequence selected from SEQ ID NO 419,420, 421, or 422. In certain such embodiments, an antisense compoundtargeted to nucleotides 92037-92155 of SEQ ID NO: 8 is selected fromIsis NO. 371665, 371669, 371671, or 171673.

In certain embodiments, a target region is nucleotides 92114-92155 ofSEQ ID NO: 8. In certain embodiments, a short antisense compound istargeted to nucleotides 92114-92155 of SEQ ID NO: 8. In certain suchembodiments, a short antisense compound targeted to nucleotides92114-92155 comprises a nucleotide sequence selected from SEQ ID NO 421or 422. In certain such embodiments, an antisense compound targeted tonucleotides 92114-92155 of SEQ ID NO: 8 is selected from Isis NO 371671or 171673.

In certain embodiments, short antisense compounds targeted to a GCCRnucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 nucleotides in length. In certain embodiments,short antisense compounds targeted to a GCCR nucleic acid are 9 to 14nucleotides in length. In certain embodiments, short antisense compoundstargeted to a GCCR nucleic acid are 10 to 14 nucleotides in length. Incertain embodiments, such short antisense compounds are short antisenseoligonucleotides.

In certain embodiments, short antisense compounds targeted to a GCCRnucleic acid are short gapmers. In certain such embodiments, shortgapmers targeted to a GCCR nucleic acid comprise at least one highaffinity modification in one or more wings of the compound. In certainembodiments, short antisense compounds targeted to a GCCR nucleic acidcomprise 1 to 3 high-affinity modifications in each wing. In certainsuch embodiments, the nucleosides or nucleotides of the wing comprise a2′ modification. In certain such embodiments, the monomers of the wingare BNA's. In certain such embodiments, the monomers of the wing areselected from α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, β-D-Methyleneoxy(4′-CH₂—O-2′) BNA, Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, Aminooxy(4′-CH₂—O—N(R)-2′) BNA and Oxyamino (4′-CH₂—N(R)—O-2′) BNA. In certainembodiments, the monomers of a wing comprise a substituent at the 2′position selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H or substituted or unsubstitutedC₁-C₁₀ alkyl. In certain embodiments, the monomers of a wing are 2′MOEnucleotides.

In certain embodiments, short antisense compounds targeted to a GCCRnucleic acid comprise a gap between the 5′ wing and the 3′ wing. Incertain embodiments the gap comprises five, six, seven, eight, nine,ten, eleven, twelve, thirteen, or fourteen monomers. In certainembodiments, the monomers of the gap are unmodifieddeoxyribonucleotides. In certain embodiments, the monomers of the gapare unmodified ribonucleotides. In certain embodiments, gapmodifications (if any) gap result in an antisense compound that, whenbound to its target nucleic acid, supports cleavage by an RNase,including, but not limited to, RNase H.

In certain embodiments, short antisense compounds targeted to a GCCRnucleic acid have uniform monomeric linkages. In certain suchembodiments, those linkages are all phosphorothioate linkages. Incertain embodiments, the linkages are all phosphodiester linkages. Incertain embodiments, short antisense compounds targeted to a GCCRnucleic acid have mixed backbones.

In certain embodiments, short antisense compounds targeted to a GCCRnucleic acid are 8 monomers in length. In certain embodiments, shortantisense compounds targeted to a GCCR nucleic acid are 9 monomers inlength. In certain embodiments, short antisense compounds targeted to aGCCR nucleic acid are 10 monomers in length. In certain embodiments,short antisense compounds targeted to a GCCR nucleic acid are 11monomers in length. In certain embodiments, short antisense compoundstargeted to a GCCR nucleic acid are monomers in length. In certainembodiments, short antisense compounds targeted to a GCCR nucleic acidare 13 monomers in length. In certain embodiments, short antisensecompounds targeted to a GCCR nucleic acid are 14 monomers in length. Incertain embodiments, short antisense compounds targeted to a GCCRnucleic acid are 15 monomers in length. In certain embodiments, shortantisense compounds targeted to a GCCR nucleic acid are 16 monomers inlength. In certain embodiments, short antisense compounds targeted to aGCCR nucleic acid comprise 9 to 15 monomers. In certain embodiments,short antisense compounds targeted to a GCCR nucleic acid comprise 10 to15 monomers. In certain embodiments, short antisense compounds targetedto a GCCR nucleic acid comprise 12 to 14 monomers. In certainembodiments, short antisense compounds targeted to a GCCR nucleic acidcomprise 12 to 14 nucleotides or nucleosides.

In certain embodiments, the invention provides methods of modulatingexpression of GCCR. In certain embodiments, such methods comprise use ofone or more short antisense compound targeted to a GCCR nucleic acid,wherein the short antisense compound targeted to a GCCR nucleic acid isfrom about 8 to about 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linkedmonomers). One of ordinary skill in the art will appreciate that thiscomprehends methods of modulating expression of GCCR using one or moreshort antisense compounds targeted to a GCCR nucleic acid of 8, 9, 10,11, 12, 13, 14, 15 or 16 monomers.

In certain embodiments, methods of modulating GCCR comprise use of ashort antisense compound targeted to a GCCR nucleic acid that is 8monomers in length. In certain embodiments, methods of modulating GCCRcomprise use of a short antisense compound targeted to a GCCR nucleicacid that is 9 monomers in length. In certain embodiments, methods ofmodulating GCCR comprise use of a short antisense compound targeted to aGCCR nucleic acid that is 10 monomers in length. In certain embodiments,methods of modulating GCCR comprise use of a short antisense compoundtargeted to a GCCR nucleic acid that is 11 monomers in length. Incertain embodiments, methods of modulating GCCR comprise use of a shortantisense compound targeted to a GCCR nucleic acid that is 12 monomersin length. In certain embodiments, methods of modulating GCCR compriseuse of a short antisense compound targeted to a GCCR nucleic acid thatis 13 monomers in length. In certain embodiments, methods of modulatingGCCR comprise use of a short antisense compound targeted to a GCCRnucleic acid that is 14 monomers in length. In certain embodiments,methods of modulating GCCR comprise use of a short antisense compoundtargeted to a GCCR nucleic acid that is 15 monomers in length. Incertain embodiments, methods of modulating GCCR comprise use of a shortantisense compound targeted to a GCCR nucleic acid that is 16 monomersin length.

In certain embodiments, methods of modulating expression of GCCRcomprise use of a short antisense compound targeted to a GCCR nucleicacid comprising 9 to 15 monomers. In certain embodiments, methods ofmodulating expression of GCCR comprise use of a short antisense compoundtargeted to a GCCR nucleic acid comprising 10 to 15 monomers. In certainembodiments, methods of modulating expression of GCCR comprise use of ashort antisense compound targeted to a GCCR nucleic acid comprising 12to 14 monomers. In certain embodiments, methods of modulating expressionof GCCR comprise use of a short antisense compound targeted to a GCCRnucleic acid comprising 12 or 14 nucleotides or nucleosides.

7. Glucagon Receiptor (GCGR)

The maintenance of normal glycemia is a carefully regulated metabolicevent. Glucagon, the 29-amino acid peptide responsible for maintainingblood glucose levels in the postabsorbative state, increases glucoserelease from the liver by activating hepatic glycogenolysis,gluconeogenesis, stimulating lipolysis in adipose tissue, andstimulating insulin secretion. During high blood glucose levels, insulinreverses the glucagon-mediated enhancement of glycogenolysis andgluconeogenesis. In patients with diabetes, insulin is either notavailable or not fully effective. While treatment for diabetes hastraditionally focused on increasing insulin levels, antagonism ofglucagon function has been considered as an alternative therapy. Asglucagon exerts its physiological effects by signaling through theglucagon receptor, the glucagon receptor has been proposed as apotential therapeutic target for diabetes (Madsen et al., Curr. Pharm.Des., 1999, 5, 683-691).

Glucagon receptor is belongs to the superfamily of G-protein-coupledreceptors having seven transmembrane domains. It is also a member of thesmaller sub-family of homologous receptors which bind peptides that arestructurally similar to glucagon. The gene encoding human glucagonreceptor was cloned in 1994 and analysis of the genomic sequencerevealed multiple introns and an 82% identity to the rat glucagonreceptor gene (Lok et al., Gene, 1994, 140, 203-209; MacNeil et al.,Biochem. Biophys. Res. Commun., 1994, 198, 328-334). Cloning of the ratglucagon receptor gene also led to the description of multiplealternative splice variants (Maget et al., FEBS Lett., 1994, 351,271-275). The human glucagon receptor gene is localized to chromosome17q25 (Menzel et al., Genomics, 1994, 20, 327-328). A missense mutationof Gly to Ser at codon 40 in the glucagon receptor gene leads to a3-fold lower affinity for glucagon (Fujisawa et al., Diabetologia, 1995,38, 983-985) and this mutation has been linked to several diseasestates, including non-insulin-dependent diabetes mellitus (Fujisawa etal., Diabetologia, 1995, 38, 983-985), hypertension (Chambers andMorris, Nat. Genet., 1996, 12, 122), and central adiposity (Siani etal., Obes. Res., 2001, 9, 722-726).

Definitions

“Glucagon receptor” is the gene product or protein of which expressionis to be modulated by administration of a short antisense compound.Glucagon receptor is generally referred to as GCGR but may also bereferred to as GR, GGR, MGC138246, MGC93090.

“GCGR nucleic acid” means any nucleic acid encoding GCGR. For example,in certain embodiments, a GCGR nucleic acid includes, withoutlimitation, a GCGR sequence encoding GCGR, an RNA sequence transcribedfrom DNA encoding GCGR, and an mRNA sequence encoding GCGR.

“GCGR mRNA” means an mRNA encoding a GCGR protein.

Therapeutic Indications

Antisense technology is an effective means for reducing glucagonreceptor (GCGR) expression and has proven to be uniquely useful in anumber of therapeutic, diagnostic, and research applications. As such,in certain embodiments, the present invention provides short antisensecompounds targeted to a nucleic acid encoding glucagon receptor, andwhich modulate the expression of glucagon receptor. Further providedherein are short antisense compounds capable of inhibiting GCGRexpression. Also provided herein are methods of treating an individualcomprising administering one or more pharmaceutical compositionscomprising a short antisense compound targeted to a GCGR nucleic acid.In certain embodiments, because short antisense compounds targeted to aGCGR nucleic acid inhibit GCGR expression, provided herein are methodsof treating a subject having a disease or condition associated with GCGRactivity by administering one or more pharmaceutical compositionscomprising a short antisense compound targeted to a GCGR nucleic acid.For example, provided herein are methods of treating a subject havinghigh blood glucose, hyperglycemia, prediabetes, diabetes, Type 2diabetes, metabolic syndrome, obesity and/or insulin resistance.

Also contemplated herein are pharmaceutical composition comprising oneor more short antisense compounds targeted to GCGR and optionally apharmaceutically acceptable carrier, diluent, enhancer or excipient.Certain compounds of the invention can also be used in the manufactureof a medicament for the treatment of diseases and disorders related toglucagon effects mediated by GCGR.

Certain embodiments of the present invention include methods of reducingthe expression of GCGR in tissues or cells comprising contacting saidcells or tissues with a short antisense compound targeted to a nucleicacid encoding GCGR or pharmaceutical composition comprising such a shortantisense compound. In certain such embodiments, the invention providesmethods of decreasing blood glucose levels, blood triglyceride levels,or blood cholesterol levels in a subject comprising administering to thesubject a short antisense compound or a pharmaceutical composition.Blood levels may be plasma levels or serum levels. Also contemplated aremethods of improving insulin sensitivity, methods of increasing GLP-1levels and methods of inhibiting hepatic glucose output in an animalcomprising administering to said animal an antisense oligonucleotide ora pharmaceutical composition of the invention. An improvement in insulinsensitivity may be indicated by a reduction in circulating insulinlevels.

In certain embodiments, the invention provides methods of treating asubject having a disease or condition associated with glucagon activityvia GCGR comprising administering to the subject a therapeutically orprophylactically effective amount of a short antisense compound or apharmaceutical composition. In certain embodiments, such disease orcondition may be a metabolic disease or condition. In certainembodiments, the metabolic disease or condition is diabetes,hyperglycemia, hyperlipidemia, metabolic syndrome X, obesity, primaryhyperglucagonemia, insulin deficiency, or insulin resistance. In someembodiments, the diabetes is Type 2 diabetes. In some embodiments theobesity is diet-induced. In some embodiments, hyperlipidemia isassociated with elevated blood lipid levels. Lipids include cholesteroland triglycerides. In one embodiment, the condition is liver steatosis.In some embodiments, the steatosis is steatohepatitis or non-alcoholicsteatohepatitis.

In certain embodiments, the invention provides methods of preventing ordelaying the onset of elevated blood glucose levels in an animal as wellas methods of preserving beta-cell function in an animal using theoligomeric compounds delineated herein.

Certain short antisense compounds targeted to GCGR can be used tomodulate the expression of GCGR in a subject in need thereof, such as ananimal, including, but not limited to, a human In certain embodiments,such methods comprise the step of administering to said animal aneffective amount of a short antisense compound that reduces expressionof GCGR RNA. In certain embodiments, short antisense compoundseffectively reduce the levels or function of GCGR RNA. Because reductionin GCGR mRNA levels can lead to alteration in GCGR protein products ofexpression as well, such resultant alterations can also be measured.Certain antisense compounds that effectively reduce the levels orfunction of GCGR RNA or protein products of expression is considered anactive antisense compound. In certain embodiments, short antisensecompounds reduce the expression of GCGR causing a reduction of RNA by atleast 10%, by at least 20%, by at least 25%, by at least 30%, by atleast 40%, by at least 50%, by at least 60%, by at least 70%, by atleast 75%, by at least 80%, by at least 85%, by at least 90%, by atleast 95%, by at least 98%, by at least 99%, or by 100%.

Further provided are methods of screening for modulators of glucagonreceptor and methods of modulating the expression of glucagon receptorin cells, tissues or animals comprising contacting said cells, tissuesor animals with one or more short antisense compounds targeted to GCGRor with compositions comprising such compounds. Methods of treating ananimal, particularly a human, suspected of having or being prone to adisease or condition associated with expression of glucagon receptor arealso set forth herein. Certain such methods comprise administering atherapeutically or prophylactically effective amount of one or more ofthe compounds or compositions of the invention to the person in need oftreatment.

The reduction of the expression of glucagon receptor may be measured,for example, in blood, plasma, serum, adipose tissue, liver or any otherbody fluid, tissue or organ of the animal. Preferably, the cellscontained within said fluids, tissues or organs being analyzed contain anucleic acid molecule encoding glucagon receptor protein and/or theglucagon receptor protein itself.

Pharmaceutical and other compositions comprising short antisensecompounds are also provided. In certain embodiments short antisensecompounds targeted to a nucleic acid encoding GCGR are utilized inpharmaceutical compositions by adding an effective amount of a compoundto a suitable pharmaceutically acceptable diluent or carrier.

The short antisense compounds targeting a GCGR nucleic acid may have anyone or more properties or characteristics of the short antisensecompounds generally described herein. In certain embodiments, shortantisense compounds targeting a GCGR nucleic acid have a motif(wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2, 2-10-1-1, 3-10-3,2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or 1-6-1. Incertain embodiments, short antisense compounds targeting a GCGR nucleicacid have a motif (wing-deoxy gap-wing) selected from 1-12-1, 2-10-2,3-10-3, 3-8-3, 1-1-10-2.

Certain Short Antisense Compounds Targeted to a GCGR Nucleic Acid

In certain embodiments, short antisense compounds are targeted to a GCGRnucleic acid having the sequence GENBANK® Accession No. NM_(—)000160.1,incorporated herein as SEQ ID NO: 9. In certain such embodiments, ashort antisense compound targeted to SEQ ID NO: 9 is at least 90%complementary to SEQ ID NO: 9. In certain such embodiments, a shortantisense compound targeted to SEQ ID NO: 9 is at least 95%complementary to SEQ ID NO: 9. In certain such embodiments, a shortantisense compound targeted to SEQ ID NO: 9 is 100% complementary to SEQID NO: 9. In certain embodiments, a short antisense compound targeted toSEQ ID NO: 9 includes a nucleotide sequence selected from the nucleotidesequences set forth in Tables 12 and 13.

The nucleotide sequences set forth in each SEQ ID NO in Tables 12 and 13are independent of any modification to a sugar moiety, aninternucleoside linkage, or a nucleobase. As such, short antisensecompounds defined by a SEQ ID NO may comprise, independently, one ormore modifications to a sugar moiety, an internucleoside linkage, or anucleobase. Short antisense compounds described by Isis Number (IsisNO.) indicate a combination of nucleobase sequence and one or moremodifications to a sugar moiety, an internucleoside linkage, or anucleobase.

In certain embodiments, short antisense compounds targeted to a GCCRnucleic acid comprise a gapmer motif. In certain embodiments, a shortantisense compound targeted to a GCCR nucleic acid comprises a 3-10-3gapmer motif. In certain embodiments, short antisense compounds targetedto a GCCR nucleic acid comprise a gapmer motif. In certain embodiments,a short antisense compound targeted to a GCCR nucleic acid comprises a3-8-3 gapmer motif. In certain embodiments, short antisense compoundstargeted to a GCCR nucleic acid comprise a gapmer motif. In certainembodiments, a short antisense compound targeted to a GCCR nucleic acidcomprises a 2-10-2 gapmer motif.

Tables 12 and 13 illustrate examples of short antisense compoundstargeted to SEQ ID NO: 9. Table 12 illustrates short antisense compoundsthat are 100% complementary to SEQ ID NO: 9. Table 13 illustrates shortantisense compounds that have one or two mismatches with respect to SEQID NO: 9. The column labeled ‘gapmer motif’ indicates the wing-gap-wingmotif of each short antisense compounds. The gap segment comprises2′-deoxynucleotides and each nucleotide of each wing segment comprises a2′-modified sugar. The particular 2′-modified sugar is also indicated inthe ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flankedby wing segments of two nucleotides, where the nucleotides of the wingsegments are 2′-MOE nucleotides. Internucleoside linkages arephosphorothioate. The short antisense compounds comprise5-methylcytidine in place of unmodified cytosine, unless “unmodifiedcytosine” is listed in the gapmer motif column, in which case theindicated cytosines are unmodified cytosines. For example, “5-mC in gaponly” indicates that the gap segment has 5-methylcytosines, while thewing segments have unmodified cytosines.

TABLE 12 Short Antisense Compounds targeted to SEQ ID NO: 9 5′ 3′ SEQISIS Target Target Gapmer ID NO. Site Site Sequence (5′-3′) Motif NO338463 378 393 TAGAGCTTCCACTTCT 3-10-3 MOE 486 338534 378 391GAGCTTCCACTTCT 3-8-3 MOE 487 327130 499 512 TGTTGGCCGTGGTA 3-8-3 MOE 488327131 500 513 ATGTTGGCCGTGGT 3-8-3 MOE 489 327132 501 514GATGTTGGCCGTGG 3-8-3 MOE 490 327133 502 515 AGATGTTGGCCGTG 3-8-3 MOE 491327134 503 516 GAGATGTTGGCCGT 3-8-3 MOE 492 327135 504 517GGAGATGTTGGCCG 3-8-3 MOE 493 327136 505 518 AGGAGATGTTGGCC 3-8-3 MOE 494327137 506 519 CAGGAGATGTTGGC 3-8-3 MOE 495 327138 507 520GCAGGAGATGTTGG 3-8-3 MOE 496 327139 508 521 GGCAGGAGATGTTG 3-8-3 MOE 497327140 531 544 GTGGTGCCAAGGCA 3-8-3 MOE 498 327141 532 545TGTGGTGCCAAGGC 3-8-3 MOE 499 327142 533 546 TTGTGGTGCCAAGG 3-8-3 MOE 500327143 534 547 TTTGTGGTGCCAAG 3-8-3 MOE 501 327144 535 548CTTTGTGGTGCCAA 3-8-3 MOE 502 327145 536 549 ACTTTGTGGTGCCA 3-8-3 MOE 503327146 537 550 CACTTTGTGGTGCC 3-8-3 MOE 504 327147 538 551GCACTTTGTGGTGC 3-8-3 MOE 505 327148 539 552 TGCACTTTGTGGTG 3-8-3 MOE 506327149 540 553 TTGCACTTTGTGGT 3-8-3 MOE 507 327150 545 558CGGTGTTGCACTTT 3-8-3 MOE 508 327151 546 559 GCGGTGTTGCACTT 3-8-3 MOE 509327152 547 560 AGCGGTGTTGCACT 3-8-3 MOE 510 327153 548 561AAGCGGTGTTGCAC 3-8-3 MOE 511 327154 549 562 GAAGCGGTGTTGCA 3-8-3 MOE 512327155 550 563 CGAAGCGGTGTTGC 3-8-3 MOE 513 327156 551 564ACGAAGCGGTGTTG 3-8-3 MOE 514 327157 552 565 CACGAAGCGGTGTT 3-8-3 MOE 515327158 553 566 ACACGAAGCGGTGT 3-8-3 MOE 516 327159 554 567AACACGAAGCGGTG 3-8-3 MOE 517 345897 684 697 GCTGCTGTACATCT 2-10-2 MOE518 327160 684 697 GCTGCTGTACATCT 3-8-3 MOE 518 327161 685 698AGCTGCTGTACATC 3-8-3 MOE 520 327162 686 699 AAGCTGCTGTACAT 3-8-3 MOE 521327163 687 700 GAAGCTGCTGTACA 3-8-3 MOE 522 327164 688 701GGAAGCTGCTGTAC 3-8-3 MOE 523 327165 689 702 TGGAAGCTGCTGTA 3-8-3 MOE 524327166 690 703 CTGGAAGCTGCTGT 3-8-3 MOE 525 327167 691 704CCTGGAAGCTGCTG 3-8-3 MOE 526 327168 692 705 ACCTGGAAGCTGCT 3-8-3 MOE 527327169 693 706 CACCTGGAAGCTGC 3-8-3 MOE 528 327170 694 707TCACCTGGAAGCTG 3-8-3 MOE 529 327171 695 708 ATCACCTGGAAGCT 3-8-3 MOE 530327172 696 709 CATCACCTGGAAGC 3-8-3 MOE 531 327173 697 710ACATCACCTGGAAG 3-8-3 MOE 532 327174 698 711 TACATCACCTGGAA 3-8-3 MOE 533327175 699 712 GTACATCACCTGGA 3-8-3 MOE 534 327176 700 713TGTACATCACCTGG 3-8-3 MOE 535 327177 701 714 GTGTACATCACCTG 3-8-3 MOE 536327178 869 882 TAGCGGGTCCTGAG 3-8-3 MOE 537 327179 870 883GTAGCGGGTCCTGA 3-8-3 MOE 538 327180 871 884 TGTAGCGGGTCCTG 3-8-3 MOE 539327181 872 885 CTGTAGCGGGTCCT 3-8-3 MOE 540 327182 873 886GCTGTAGCGGGTCC 3-8-3 MOE 541 327183 874 887 GGCTGTAGCGGGTC 3-8-3 MOE 542327184 875 888 TGGCTGTAGCGGGT 3-8-3 MOE 543 327185 876 889CTGGCTGTAGCGGG 3-8-3 MOE 544 327186 877 890 TCTGGCTGTAGCGG 3-8-3 MOE 545327187 878 891 TTCTGGCTGTAGCG 3-8-3 MOE 546 327188 955 968TGAACACCGCGGCC 3-8-3 MOE 547 327189 956 969 ATGAACACCGCGGC 3-8-3 MOE 548327190 957 970 CATGAACACCGCGG 3-8-3 MOE 549 327191 958 971GCATGAACACCGCG 3-8-3 MOE 550 327192 959 972 TGCATGAACACCGC 3-8-3 MOE 551327193 960 973 TTGCATGAACACCG 3-8-3 MOE 552 327194 961 974ATTGCATGAACACC 3-8-3 MOE 553 327195 962 975 TATTGCATGAACAC 3-8-3 MOE 554327196 963 976 ATATTGCATGAACA 3-8-3 MOE 555 327197 964 977CATATTGCATGAAC 3-8-3 MOE 556 327198 1019 1032 AGGTTGTGCAGGTA 3-8-3 MOE557 327199 1020 1033 CAGGTTGTGCAGGT 3-8-3 MOE 558 327200 1021 1034GCAGGTTGTGCAGG 3-8-3 MOE 559 327201 1022 1035 AGCAGGTTGTGCAG 3-8-3 MOE560 327202 1023 1036 CAGCAGGTTGTGCA 3-8-3 MOE 561 327203 1024 1037CCAGCAGGTTGTGC 3-8-3 MOE 562 327204 1025 1038 CCCAGCAGGTTGTG 3-8-3 MOE563 327205 1026 1039 GCCCAGCAGGTTGT 3-8-3 MOE 564 327206 1027 1040GGCCCAGCAGGTTG 3-8-3 MOE 565 327207 1028 1041 AGGCCCAGCAGGTT 3-8-3 MOE566 338491 1160 1175 TGTCATTGCTGGTCCA 3-10-3 MOE 567 338562 1160 1173TCATTGCTGGTCCA 3-8-3 MOE 568 338498 1307 1322 TGGCCAGCCGGAACTT 3-10-3MOE 569 338569 1307 1320 GCCAGCCGGAACTT 3-8-3 MOE 570 338499 1329 1344GGGATGAGGGTCAGCG 3-10-3 MOE 571 338570 1329 1342 GATGAGGGTCAGCG 3-8-3MOE 572 385067 1364 1377 AAGGCAAAGACCAC 3-8-3 MOE 573 338573 1401 1414GGAGCGCAGGGTGC 3-8-3 MOE 574 338580 1487 1500 TGCACCTCCTTGTT 3-8-3 MOE575

TABLE 13 Short antisense compounds targeted to SEQ ID NO: 1 and having 1or 2 mismatches 5′ 3′ SEQ ISIS Target Target Gapmer ID NO. Site SiteSequence (5′-3′) Motif NO 338577 158 171 CAGCAGACCCTGGA 3-8-3 MOE 576338458 237 252 ACATCTGGCAGAGGTT 3-10-3 MOE 577 338529 237 250ATCTGGCAGAGGTT 3-8-3 MOE 578 338466 318 333 CAGGCCAGCAGGAGTA 3-10-3 MOE579 338537 318 331 GGCCAGCAGGAGTA 3-8-3 MOE 580 338533 364 377CAAACAAAAAGTCC 3-8-3 MOE 582 338462 364 379 CTCAAACAAAAAGTCC 3-10-3 MOE581 338535 397 410 GGTGACATTGGTCA 3-8-3 MOE 584 338464 397 412GTGGTGACATTGGTCA 3-10-3 MOE 583 338466 470 485 CAGGCCAGCAGGAGTA 3-10-3MOE 579 338537 470 483 GGCCAGCAGGAGTA 3-8-3 MOE 580 385048 497 510TTGGCAGTGGTGTT 3-8-3 MOE 587 385049 500 513 ATGTTGGCAGTGGT 3-8-3 MOE 588338467 503 518 AGGAAATGTTGGCAGT 3-10-3 MOE 589 338538 503 516GAAATGTTGGCAGT 3-8-3 MOE 590 385050 506 519 CAGGAAATGTTGGC 3-8-3 MOE 591385051 509 522 GGGCAGGAAATGTT 3-8-3 MOE 592 385052 523 536AAGGTAGGTACCAG 3-8-3 MOE 593 385053 526 539 ACCAAGGTAGGTAC 3-8-3 MOE 594385056 535 548 CTTTGTGGCACCAA 3-8-3 MOE 595 385057 538 551GCACTTTGTGGCAC 3-8-3 MOE 596 338539 539 552 TGCACTTTGTGGCA 3-8-3 MOE 597385058 541 554 GCTGCACTTTGTGG 3-8-3 MOE 598 385059 544 557GGTGCTGCACTTTG 3-8-3 MOE 599 385060 547 560 GGCGGTGCTGCACT 3-8-3 MOE 600385063 556 569 TGAACACTAGGCGG 3-8-3 MOE 601 385064 559 572TCTTGAACACTAGG 3-8-3 MOE 602 338469 561 576 CACCTCTTGAACACTA 3-10-3 MOE603 338540 561 574 CCTCTTGAACACTA 3-8-3 MOE 604 385065 562 575ACCTCTTGAACACT 3-8-3 MOE 605 385066 565 578 CACACCTCTTGAAC 3-8-3 MOE 606338541 590 603 CCTCGAACCCACTG 3-8-3 MOE 607 338473 658 673CTTCTGGACCTCGATC 3-10-3 MOE 608 338544 658 671 TCTGGACCTCGATC 3-8-3 MOE609 338474 681 696 CTGCTATACATCTTGG 3-10-3 MOE 610 338545 681 694GCTATACATCTTGG 3-8-3 MOE 611 338475 703 718 CACGGTGTACATCACC 3-10-3 MOE612 338546 703 716 CGGTGTACATCACC 3-8-3 MOE 613 338547 718 731ACAGACTGTAGCCC 3-8-3 MOE 615 338476 718 733 GGACAGACTGTAGCCC 3-10-3 MOE614 338550 889 902 CATCGCCAATCTTC 3-8-3 MOE 617 338479 889 904GTCATCGCCAATCTTC 3-10-3 MOE 616 338551 899 912 ACACTGAGGTCATC 3-8-3 MOE619 338480 899 914 TCACACTGAGGTCATC 3-10-3 MOE 618 338552 924 937CGCCCCGTCACTGA 3-8-3 MOE 620 338555 992 1005 AGCAACCAGCAATA 3-8-3 MOE622 338484 992 1007 CCAGCAACCAGCAATA 3-10-3 MOE 621 338485 1018 1033CAGGCTGTACAGGTAC 3-10-3 MOE 623 338556 1018 1031 GGCTGTACAGGTAC 3-8-3MOE 624 338558 1051 1064 AGCTCCTCTCAGAG 3-8-3 MOE 626 338487 1051 1066GAAGCTCCTCTCAGAG 3-10-3 MOE 625 338559 1079 1092 CAGCCAATGCCCAG 3-8-3MOE 628 338488 1079 1094 CCCAGCCAATGCCCAG 3-10-3 MOE 627 338560 11311144 AAACAGACACTTGA 3-8-3 MOE 630 338489 1131 1146 TCAAACAGACACTTGA3-10-3 MOE 629 338490 1145 1160 AGCACTGAACATTCTC 3-10-3 MOE 631 3385611145 1158 CACTGAACATTCTC 3-8-3 MOE 632 338563 1181 1194 ATCCACCAGAATCC3-8-3 MOE 634 338492 1181 1196 GGATCCACCAGAATCC 3-10-3 MOE 633 3385641216 1229 TGATCAGTAAGGCC 3-8-3 MOE 635 338565 1232 1245 ACAAAGATGAAAAA3-8-3 MOE 637 338494 1232 1247 GGACAAAGATGAAAAA 3-10-3 MOE 636 3385661267 1280 CACGCAGCTTGGCC 3-8-3 MOE 639 338495 1267 1282 GGCACGCAGCTTGGCC3-10-3 MOE 638 338571 1344 1357 GACCCCCAGCAGAG 3-8-3 MOE 641 338500 13441359 TGGACCCCCAGCAGAG 3-10-3 MOE 640 385068 1366 1379 CAAAGGCAAAGACC3-8-3 MOE 642 385069 1369 1382 TCACAAAGGCAAAG 3-8-3 MOE 643 385070 13721385 CAGTCACAAAGGCA 3-8-3 MOE 644 385071 1375 1388 CGTCAGTCACAAAG 3-8-3MOE 645 385072 1378 1391 GCTCGTCAGTCACA 3-8-3 MOE 646 385073 1381 1394CATGCTCGTCAGTC 3-8-3 MOE 647 386608 1384 1397 GGGCATGCTCGTCA 1-12-1 MOE648 386593 1384 1397 GGGCATGCTCGTCA 2-10-2 MOE 648 396146 1384 1397GGGCATGCTCGTCA 2-10-2 MOE 648 338572 1384 1397 GGGCATGCTCGTCA 3-8-3 MOE648 396149 1384 1397 GGGCATGCTCGTCA 1-1-10-2 648 2′-(butyl- acetamido)-palmit- amide/OMe/ OMe 386627 1384 1397 GGGCATGCTCGTCA 2-10-2 648Methylene- oxy BNA 386610 1387 1400 CTTGGGCATGCTCG 1-12-1 MOE 654 3865951387 1400 CTTGGGCATGCTCG 2-10-2 MOE 654 385074 1387 1400 CTTGGGCATGCTCG3-8-3 MOE 654 385075 1390 1403 TGCCTTGGGCATGC 3-8-3 MOE 657 385076 13931406 GGGTGCCTTGGGCA 3-8-3 MOE 648 385077 1396 1409 GCAGGGTGCCTTGG 3-8-3MOE 659 385078 1399 1412 AGCGCAGGGTGCCT 3-8-3 MOE 660 338502 1401 1416GTGGAGCGCAGGGTGC 3-10-3 MOE 661 385079 1402 1415 TGGAGCGCAGGGTG 3-8-3MOE 662 385080 1405 1418 TGGTGGAGCGCAGG 3-8-3 MOE 663 385081 1408 1421GCTTGGTGGAGCGC 3-8-3 MOE 664 385082 1411 1424 AGAGCTTGGTGGAG 3-8-3 MOE665 338503 1412 1427 AAAAGAGCTTGGTGGA 3-10-3 MOE 666 338574 1412 1425AAGAGCTTGGTGGA 3-8-3 MOE 667 385083 1414 1427 AAAAGAGCTTGGTG 3-8-3 MOE668 385084 1417 1430 CAAAAAAGAGCTTG 3-8-3 MOE 669 338504 1434 1449AAGGAGCTGAGGAACA 3-10-3 MOE 670 338575 1434 1447 GGAGCTGAGGAACA 3-8-3MOE 671 327167 1441 1454 CCTGGAAGCTGCTG 3-8-3 MOE 526 338576 1445 1458AGACCCTGGAAGGA 3-8-3 MOE 673 338505 1445 1460 GCAGACCCTGGAAGGA 3-10-3MOE 672 338506 1449 1464 ACCAGCAGACCCTGGA 3-10-3 MOE 674 338577 14491462 CAGCAGACCCTGGA 3-8-3 MOE 576 338507 1464 1479 CAGTAGAGAACAGCCA3-10-3 MOE 676 338578 1464 1477 GTAGAGAACAGCCA 3-8-3 MOE 677 338508 14751490 TGTTGAGGAAACAGTA 3-10-3 MOE 678 338579 1475 1488 TTGAGGAAACAGTA3-8-3 MOE 679 338509 1487 1502 CCTGCACCTCCTTGTT 3-10-3 MOE 680 3385801610 1623 TGCACCTCCTTGTT 3-8-3 MOE 575

In certain embodiments, a target region is nucleotides 378-391 of SEQ IDNO: 9. In certain embodiments, a short antisense compound is targeted tonucleotides 378-391 of SEQ ID NO: 9. In certain such embodiments, ashort antisense compound targeted to nucleotides 378-391 comprises anucleotide sequence selected from SEQ ID NO 486 or 487. In certain suchembodiments, a short antisense compound targeted to nucleotides 378-391of SEQ ID NO: 9 is selected from Isis No 338463 or 338534.

In certain embodiments, a target region is nucleotides 499-521 of SEQ IDNO: 9. In certain embodiments, a short antisense compound is targeted tonucleotides 499-521 of SEQ ID NO: 9. In certain such embodiments, ashort antisense compound targeted to nucleotides 499-521 comprises anucleotide sequence selected from SEQ ID NO 488, 489, 490, 491, 492,493, 494, 495, 496, or 497. In certain such embodiments, a shortantisense compound targeted to nucleotides 499-521 of SEQ ID NO: 9 isselected from Isis No 327130, 327131, 327132, 327133, 327134, 327135,327136, 327137, 327138, or 327139.

In certain embodiments, a target region is nucleotides 531-553 of SEQ IDNO: 9. In certain embodiments, a short antisense compound is targeted tonucleotides 531-553 of SEQ ID NO: 9. In certain such embodiments, ashort antisense compound targeted to nucleotides 531-553 comprises anucleotide sequence selected from SEQ ID NO 498, 499, 500, 501, 502,503, 504, 505, 506, or 507. In certain such embodiments, a shortantisense compound targeted to nucleotides 531-553 of SEQ ID NO: 9 isselected from Isis No 327140, 327141, 327142, 327143, 327144, 327145,327146, 327147, 327148, or 327149.

In certain embodiments, a target region is nucleotides 545-567 of SEQ IDNO: 9. In certain embodiments, a short antisense compound is targeted tonucleotides 545-567 of SEQ ID NO: 9. In certain such embodiments, ashort antisense compound targeted to nucleotides 545-567 comprises anucleotide sequence selected from SEQ ID NO 508, 509, 510, 511, 512,513, 514, 515, 516, or 517. In certain such embodiments, a shortantisense compound targeted to nucleotides 545-567 of SEQ ID NO: 9 isselected from Isis No 327150, 327151, 327152, 327153, 327154, 327155,327156, 327157, 327158, or 327159.

In certain embodiments, a target region is nucleotides 531-567 of SEQ IDNO: 9. In certain embodiments, a short antisense compound is targeted tonucleotides 531-567 of SEQ ID NO: 9. In certain such embodiments, ashort antisense compound targeted to nucleotides 531-567 comprises anucleotide sequence selected from SEQ ID NO 498, 499, 500, 501, 502,503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, or517. In certain such embodiments, a short antisense compound targeted tonucleotides 531-567 of SEQ ID NO: 9 is selected from Isis No 327140,327141, 327142, 327143, 327144, 327145, 327146, 327147, 327148, 327149,327150, 327151, 327152, 327153, 327154, 327155, 327156, 327157, 327158,or 327159.

In certain embodiments, a target region is nucleotides 684-714 of SEQ IDNO: 9. In certain embodiments, a short antisense compound is targeted tonucleotides 684-714 of SEQ ID NO: 9. In certain such embodiments, ashort antisense compound targeted to nucleotides 684-714 comprises anucleotide sequence selected from SEQ ID NO 518, 520, 521, 522, 523,524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, or 536. Incertain such embodiments, a short antisense compound targeted tonucleotides 684-714 of SEQ ID NO: 9 is selected from Isis No 345897,327160, 327161, 327162, 327163, 327164, 327165, 327166, 327167, 327168,327169, 327170, 327171, 327172, 327173, 327174, 327175, 327176, or327177.

In certain embodiments, a target region is nucleotides 869-891 of SEQ IDNO: 9. In certain embodiments, a short antisense compound is targeted tonucleotides 869-891 of SEQ ID NO: 9. In certain such embodiments, ashort antisense compound targeted to nucleotides 869-891 comprises anucleotide sequence selected from SEQ ID NO 537, 538, 539, 540, 541,542, 543, 544, 545, or 546. In certain such embodiments, a shortantisense compound targeted to nucleotides 869-891 of SEQ ID NO: 9 isselected from Isis No 327178, 327179, 327180, 327181, 327182, 327183,327184, 327185, 327186, or 327187.

In certain embodiments, a target region is nucleotides 955-977 of SEQ IDNO: 9. In certain embodiments, a short antisense compound is targeted tonucleotides 955-977 of SEQ ID NO: 9. In certain such embodiments, ashort antisense compound targeted to nucleotides 955-977 comprises anucleotide sequence selected from SEQ ID NO 547, 548, 549, 550, 551,552, 553, 554, 555, or 556. In certain such embodiments, a shortantisense compound targeted to nucleotides 955-977 of SEQ ID NO: 9 isselected from Isis No 327188, 327189, 327190, 327191, 327192, 327193,327194, 327195, 327196, or 327197.

In certain embodiments, a target region is nucleotides 1019-1041 of SEQID NO: 9. In certain embodiments, a short antisense compound is targetedto nucleotides 1019-1041 of SEQ ID NO: 9. In certain such embodiments, ashort antisense compound targeted to nucleotides 1019-1041 comprises anucleotide sequence selected from SEQ ID NO 557, 558, 559, 560, 561,562, 563, 564, 565, or 566. In certain such embodiments, a shortantisense compound targeted to nucleotides 1019-1041 of SEQ ID NO: 9 isselected from Isis No 327198, 327199, 327200, 327201, 327202, 327203,327204, 327205, 327206, or 327207.

In certain embodiments, a target region is nucleotides 1160-1175 of SEQID NO: 9. In certain embodiments, a short antisense compound is targetedto nucleotides 1160-1175 of SEQ ID NO: 9. In certain such embodiments, ashort antisense compound targeted to nucleotides 1160-1175 comprises anucleotide sequence selected from SEQ ID NO 567 or 568. In certain suchembodiments, a short antisense compound targeted to nucleotides1160-1175 of SEQ ID NO: 9 is selected from Isis No 338491 or 338562.

In certain embodiments, a target region is nucleotides 1307-1377 of SEQID NO: 9. In certain embodiments, a short antisense compound is targetedto nucleotides 1307-1377 of SEQ ID NO: 9. In certain such embodiments, ashort antisense compound targeted to nucleotides 1307-1377 comprises anucleotide sequence selected from SEQ ID NO 569, 570, 571, 572, or 573.In certain such embodiments, a short antisense compound targeted tonucleotides 1307-1377 of SEQ ID NO: 9 is selected from Isis No 338498,338569, 338499, 338570, or 385067.

In certain embodiments, a target region is nucleotides 1307-1414 of SEQID NO: 9. In certain embodiments, a short antisense compound is targetedto nucleotides 1307-1414 of SEQ ID NO: 9. In certain such embodiments, ashort antisense compound targeted to nucleotides 1307-1414 comprises anucleotide sequence selected from SEQ ID NO 569, 570, 571, 572, 573, or574. In certain such embodiments, a short antisense compound targeted tonucleotides 1307-1414 of SEQ ID NO: 9 is selected from Isis No 338498,338569, 338499, 338570, 385067, or 338573.

In certain embodiments, short antisense compounds targeted to a GCGRnucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 nucleotides in length. In certain embodiments,short antisense compounds targeted to a GCGR nucleic acid are 9 to 14nucleotides in length. In certain embodiments, short antisense compoundstargeted to a GCGR nucleic acid are 10 to 14 nucleotides in length. Incertain embodiments, such short antisense compounds are short antisenseoligonucleotides.

In certain embodiments, short antisense compounds targeted to a GCGRnucleic acid are short gapmers. In certain such embodiments, shortgapmers targeted to a GCGR nucleic acid comprise at least one highaffinity modification in one or more wings of the compound. In certainembodiments, short antisense compounds targeted to a GCGR nucleic acidcomprise 1 to 3 high-affinity modifications in each wing. In certainsuch embodiments, the nucleosides or nucleotides of the wing comprise a2′ modification. In certain such embodiments, the monomers of the wingare BNA's. In certain such embodiments, the monomers of the wing areselected from α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, β-D-Methyleneoxy(4′-CH₂—O-2′) BNA, Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, Aminooxy(4′-CH₂—O—N(R)-2′) BNA and Oxyamino (4′-CH₂—N(R)—O-2′) BNA. In certainembodiments, the monomers of a wing comprise a substituent at the 2′position selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H or substituted or unsubstitutedC₁-C₁₀ alkyl. In certain embodiments, the monomers of a wing are 2′MOEnucleotides.

In certain embodiments, short antisense compounds targeted to a GCGRnucleic acid comprise a gap between the 5′ wing and the 3′ wing. Incertain embodiments the gap comprises five, six, seven, eight, nine,ten, eleven, twelve, thirteen, or fourteen monomers. In certainembodiments, the monomers of the gap are unmodifieddeoxyribonucleotides. In certain embodiments, the monomers of the gapare unmodified ribonucleotides. In certain embodiments, gapmodifications (if any) gap result in an antisense compound that, whenbound to its target nucleic acid, supports cleavage by an RNase,including, but not limited to, RNase H.

In certain embodiments, short antisense compounds targeted to a GCGRnucleic acid have uniform monomeric linkages. In certain suchembodiments, those linkages are all phosphorothioate linkages. Incertain embodiments, the linkages are all phosphodiester linkages. Incertain embodiments, short antisense compounds targeted to a GCGRnucleic acid have mixed backbones.

In certain embodiments, short antisense compounds targeted to a GCGRnucleic acid are 8 monomers in length. In certain embodiments, shortantisense compounds targeted to a GCGR nucleic acid are 9 monomers inlength. In certain embodiments, short antisense compounds targeted to aGCGR nucleic acid are 10 monomers in length. In certain embodiments,short antisense compounds targeted to a GCGR nucleic acid are 11monomers in length. In certain embodiments, short antisense compoundstargeted to a GCGR nucleic acid are monomers in length. In certainembodiments, short antisense compounds targeted to a GCGR nucleic acidare 13 monomers in length. In certain embodiments, short antisensecompounds targeted to a GCGR nucleic acid are 14 monomers in length. Incertain embodiments, short antisense compounds targeted to a GCGRnucleic acid are 15 monomers in length. In certain embodiments, shortantisense compounds targeted to a GCGR nucleic acid are 16 monomers inlength. In certain embodiments, short antisense compounds targeted to aGCGR nucleic acid comprise 9 to 15 monomers. In certain embodiments,short antisense compounds targeted to a GCGR nucleic acid comprise 10 to15 monomers. In certain embodiments, short antisense compounds targetedto a GCGR nucleic acid comprise 12 to 14 monomers. In certainembodiments, short antisense compounds targeted to a GCGR nucleic acidcomprise 12 to 14 nucleotides or nucleosides.

In certain embodiments, the invention provides methods of modulatingexpression of GCGR. In certain embodiments, such methods comprise use ofone or more short antisense compound targeted to a GCGR nucleic acid,wherein the short antisense compound targeted to a GCGR nucleic acid isfrom about 8 to about 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linkedmonomers). One of ordinary skill in the art will appreciate that thiscomprehends methods of modulating expression of GCGR using one or moreshort antisense compounds targeted to a GCGR nucleic acid of 8, 9, 10,11, 12, 13, 14, 15 or 16 monomers.

In certain embodiments, methods of modulating GCGR comprise use of ashort antisense compound targeted to a GCGR nucleic acid that is 8monomers in length. In certain embodiments, methods of modulating GCGRcomprise use of a short antisense compound targeted to a GCGR nucleicacid that is 9 monomers in length. In certain embodiments, methods ofmodulating GCGR comprise use of a short antisense compound targeted to aGCGR nucleic acid that is 10 monomers in length. In certain embodiments,methods of modulating GCGR comprise use of a short antisense compoundtargeted to a GCGR nucleic acid that is 11 monomers in length. Incertain embodiments, methods of modulating GCGR comprise use of a shortantisense compound targeted to a GCGR nucleic acid that is 12 monomersin length. In certain embodiments, methods of modulating GCGR compriseuse of a short antisense compound targeted to a GCGR nucleic acid thatis 13 monomers in length. In certain embodiments, methods of modulatingGCGR comprise use of a short antisense compound targeted to a GCGRnucleic acid that is 14 monomers in length. In certain embodiments,methods of modulating GCGR comprise use of a short antisense compoundtargeted to a GCGR nucleic acid that is 15 monomers in length. Incertain embodiments, methods of modulating GCGR comprise use of a shortantisense compound targeted to a GCGR nucleic acid that is 16 monomersin length.

In certain embodiments, methods of modulating expression of GCGRcomprise use of a short antisense compound targeted to a GCGR nucleicacid comprising 9 to 15 monomers. In certain embodiments, methods ofmodulating expression of GCGR comprise use of a short antisense compoundtargeted to a GCGR nucleic acid comprising 10 to 15 monomers. In certainembodiments, methods of modulating expression of GCGR comprise use of ashort antisense compound targeted to a GCGR nucleic acid comprising 12to 14 monomers. In certain embodiments, methods of modulating expressionof GCGR comprise use of a short antisense compound targeted to a GCGRnucleic acid comprising 12 or 14 nucleotides or nucleosides.

8. DGAT2

Diacylglycerol transferase 2 (also known as DGAT2, diacylglycerolO-transferase 2, acyl-CoA:diacylglycerol acyltransferase 2),Diacylglycerol transferase 2 has been shown to be implicated in theabsorption process of triglycerides (also called triacylglycerols) fromfood.

The absorption of triglycerides from food is a very efficient processwhich occurs by a series of steps wherein the dietary triacylglycerolsare hydrolyzed in the intestinal lumen and then resynthesized withinenterocytes. The resynthesis of triacylglycerols can occur via themonoacylglycerol pathway which commences with monoacylglycerolacyltransferase (MGAT) catalyzing the synthesis of diacylglycerol frommonoacylglycerol and fatty acyl-CoA. An alternative synthesis ofdiacylglycerols is provided by the glycerol-phosphate pathway whichdescribes the coupling of two molecules of fatty acyl-CoA toglycerol-3-phosphate. In either case, diacylglycerol is then acylatedwith another molecule of fatty acyl-CoA in a reaction catalyzed by oneof two diacylglycerol acyltransferase enzymes to form the triglyceride(Farese et al., Curr. Opin. Lipidol., 2000, 11, 229-234).

The reaction catalyzed by diacylglycerol acyltransferase is the finaland only committed step in triglyceride synthesis. As such,diacylglycerol acyltransferase is involved in intestinal fat absorption,lipoprotein assembly, regulating plasma triglyceride concentrations, andfat storage in adipocytes. The first diacylglycerol acyltransferase,diacylglycerol transferase 1, was identified in 1960 and the human andmouse genes encoding this protein were isolated in 1998 (Cases et al.,Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 13018-13023; Oelkers et al., J.Biol. Chem., 1998, 273, 26765-26771). Mice lacking diacylglycerolacyltransferase 1 are viable and can still synthesize triglyceridesthrough other biological routes, suggesting the existence of multiplemechanisms for triglyceride synthesis (Smith et al., Nat. Genet., 2000,25, 87-90).

A second diacylglycerol transferase, diacylglycerol transferase 2 (alsoknown as DGAT2, diacylglycerol O-transferase 2, acyl-CoA:diacylglycerolacyltransferase 2), was subsequently identified in the fungusMortierella, humans and mice (Cases et al., J. Biol. Chem., 2001, 276,38870-38876; Lardizabal et al., J. Biol. Chem., 2001, 276, 38862-38869).Enzymatic assays indicate that this recently identified protein doespossess diacylglycerol transferase activity that utilizes a broad rangeof long chain fatty acyl-CoA substrates (Cases et al., J. Biol. Chem.,2001, 276, 38870-38876).

Diacylglycerol transferase 2 is a member of a family of genes whosesequences are unrelated to diacylglycerol transferase 1. In addition todiffering in sequence compared to diacylglycerol transferase 1, in vitroassays illustrate that diacylglycerol transferase 2 has higher activityat lower concentrations of magnesium chloride and oleoyl-CoA (Cases etal., J. Biol. Chem., 2001, 276, 38870-38876). The predicted proteinsequence of diacylglycerol transferase 2 contains at least one putativetransmembrane domain, three potential N-linked glycosylation sites, sixpotential protein kinase C phosphorylation consensus sites, as well assequences in common with a putative glycerol phosphorylation site foundin acyltransferase enzymes (Cases et al., J. Biol. Chem., 2001, 276,38870-38876). The International Radiation Hybrid Mapping Consortium hasmapped human diacylglycerol transferase 2 to chromosome 11q13.3.

In human tissues, the highest levels of diacylglycerol transferase 2 aredetected in liver and white adipose tissues, with lower levels found inmammary gland, testis and peripheral blood leukocytes (Cases et al., J.Biol. Chem., 2001, 276, 38870-38876). Two mRNA species of 2.4 and 1.8kilobases are detected in human tissues, whereas the majordiacylglycerol transferase 2 mRNA species in mouse tissues is 2.4kilobases. In addition to liver and white adipose tissues,diacylglycerol transferase 2 is expressed in all segments of the smallintestine in mice, with higher expression in the proximal intestine andlower expression in the distal intestine (Cases et al., J. Biol. Chem.,2001, 276, 38870-38876).

Diacylglycerol transferase activity exhibits distinct patterns duringpostnatal development of the rat liver. As there is no correlationbetween the mRNA expression and activity patterns, post-translationalmodifications may participate in the regulation of diacylglyceroltransferase 2 activity during rat development (Waterman et al., J.Lipid. Res., 2002, 43, 1555-1562).

Diacylglycerol transferase 2 mRNA is preferentially upregulated byinsulin treatment, as shown by in vitro assays measuring thediacylglycerol activity from the membrane fraction of cultured mouseadipocytes (Meegalla et al., Biochem. Biophys. Res. Commun., 2002, 298,317-323). In fasting mice, diacylglycerol transferase 2 expression isgreatly reduced, and dramatically increases upon refeeding. Theexpression patterns of two enzymes that participate in fatty acidsynthesis, acetyl-CoA carboxylase and fatty acid synthase, respond tofasting and refeeding in a similar fashion. These results, combined withthe observation that diacylglycerol transferase 2 is abundantlyexpressed in liver, suggest that diacylglycerol transferase 2 is tightlylinked to the endogenous fatty acid synthesis pathway (Meegalla et al.,Biochem. Biophys. Res. Commun., 2002, 298, 317-323).

Studies of mice harboring a disruption in the diacylglycerolacyltransferase 1 gene provide evidence that diacylglycerolacyltransferase 2 contributes to triglyceride synthesis. Levels ofdiacylglycerol transferase 2 mRNA expression are similar in intestinalsegments from both wild type and diacylglycerol transferase 1-deficientmice (Buhman et al., J. Biol. Chem., 2002, 277, 25474-25479). Usingmagnesium chloride to distinguish between diacylglycerol transferase 1and 2 activity, Buhman, et al. observed that, in diacylglyceroltransferase 1-deficient mice, diacylglycerol transferase activity isreduced to 50% in the proximal intestine and to 10-15% in the distalintestine (Buhman et al., J. Biol. Chem., 2002, 277, 25474-25479).

Additionally, diacylglycerol transferase 2 mRNA levels are notup-regulated the liver or adipose tissues of diacylglycerol transferase1-deficient mice, even after weeks of high-fat diet (Cases et al., J.Biol. Chem., 2001, 276, 38870-38876; Chen et al., J. Clin. Invest.,2002, 109, 1049-1055). However, in ob/ob mice, which have a mutation inthe leptin gene that results in obesity, diacylglycerol transferase 2 ismore highly expressed than in wild type mice, suggesting thatdiacylglycerol transferase 2 may be partly responsible for the highlyaccumulated fat mass seen in these mice. Furthermore, the combinedmutations of leptin and diacylglycerol transferase 1 leads to athree-fold elevation in diacylglycerol transferase 2 expression in whiteadipose tissue, compared to the levels in the same tissue fromdiacylglycerol transferase 1-deficient mice (Chen et al., J. Clin.Invest., 2002, 109, 1049-1055). Diacylglycerol transferase 2 mRNA isalso upregulated in the skin of these mice (Chen et al., J. Clin.Invest., 2002, 109, 175-181). These data suggest leptin normallydownregulates diacylglycerol transferase 2 expression, and that theupregulation of diacylglycerol transferase 2 in white adipose tissue inthese mice may provide an alternate pathway for the triglyceridesynthesis that still occurs in leptin deficient/diacylglyceroltransferase 1-deficient mice (Chen et al., J. Clin. Invest., 2002, 109,1049-1055).

Diacylglycerol acyltransferase 1 knockout mice exhibit interestingphenotypes in that they are lean, resistant to diet-induce obesity, havedecreased levels of tissue triglycerides and increased sensitivity toinsulin and leptin (Chen et al., J. Clin. Invest., 2002, 109, 1049-1055;Smith et al., Nat. Genet., 2000, 25, 87-90). As diacylglyceroltransferase 2 also participates in triglyceride synthesis, interferingwith diacylglycerol transferase 2 may similarly lead to reduced body fatcontent.

Definitions

“DGAT2” means the gene product or protein of which expression is to bemodulated by administration of a short antisense compound.

“DGAT2 nucleic acid” means any nucleic acid encoding DGAT2. For example,in certain embodiments, a DGAT2 nucleic acid includes, withoutlimitation, a DNA sequence encoding DGAT2, an RNA sequence transcribedfrom DNA encoding DGAT2, and an mRNA sequence encoding DGAT2.

“DGAT2 mRNA” means an mRNA encoding DGAT2.

Therapeutic Indications

Antisense technology is an effective means for reducing DGAT2 expressionand has proven to be uniquely useful in a number of therapeutic,diagnostic, and research applications. As such, in certain embodiments,the present invention provides compounds targeted to nucleic acidencoding DGAT2, which modulate the expression of DGAT2. Further providedherein are short antisense compounds capable of effectively inhibitingDGAT2 expression.

In certain embodiments, a subject, suspected of having a disease orassociated with DGAT2 is treated by administering one or more shortantisense compounds targeted to a nucleic acid encoding DGAT2. Forexample, in a non-limiting embodiment, such methods comprise the step ofadministering to an animal a therapeutically effective amount of a shortantisense compound. In certain such embodiments, short antisensecompounds effectively inhibit the activity of DGAT2 or inhibit theexpression of DGAT2. In one embodiment, the activity or expression ofDGAT2 in a subject is inhibited by at least 10%, by at least 20%, by atleast 25%, by at least 30%, by at least 40%, by at least 50%, by atleast 60%, by at least 70%, by at least 75%, by at least 80%, by atleast 85%, by at least 90%, by at least 95%, by at least 98%, by atleast 99%, or by 100%. In certain embodiments, the activity orexpression of DGAT2 in a subject is inhibited by about 30%. Morepreferably, the activity or expression of DGAT2 in a subject isinhibited by 50% or more.

The reduction of the expression of DGAT2 may be measured, for example,in blood, plasma, serum, adipose tissue, liver or any other body fluid,tissue or organ of the animal. Preferably, the cells contained withinsaid fluids, tissues or organs being analyzed contain a nucleic acidmolecule encoding DGAT2 and/or the DGAT2 protein itself.

In certain embodiments, pharmaceutical and other compositions comprisingthe compounds of the invention are also provided. For example, shortantisense compounds targeted to a DGAT2 nucleic acid can be utilized inpharmaceutical compositions by adding an effective amount of a compoundto a suitable pharmaceutically acceptable diluent or carrier.

Certain short antisense compounds targeting DGAT2 may have any one ormore properties or characteristics of the short antisense compoundsgenerally described herein. In certain embodiments, short antisensecompounds targeting a DGAT2 nucleic acid have a motif (wing-deoxygap-wing) selected from 1-12-1, 1-1-10-2, 2-10-1-1, 3-10-3, 2-10-3,2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or 1-6-1. In certainembodiments, short antisense compounds targeting a DGAT2 nucleic acidhave a motif (wing-deoxy gap-wing) selected from 1-10-1, 2-10-2 and3-10-3.

Provided herein are methods of treating an individual by administeringone or more short antisense compound targeted to a DGAT2 nucleic acid ora pharmaceutical composition comprising such compound. Further providedare methods of treating a subject having a disease or conditionsassociated with DGAT2 activity by administering a short antisensecompound targeted to a DGAT2 nucleic acid. Diseases and conditionsassociated with DGAT2 include, but are not limited to, cardiovasculardisorders, obesity, diabetes, cholesterolemia, and liver steatosis.

Certain Short Antisense Compounds Targeted to a DGAT2 Nucleic Acid

In certain embodiments, short antisense compounds are targeted to aDGAT2 nucleic acid having the sequence of GENBANK® Accession No.NM_(—)032564.2, incorporated herein as SEQ ID NO: 10. In certain suchembodiments, a short antisense compound targeted to SEQ ID NO: 10 is atleast 90% complementary to SEQ ID NO: 10. In certain such embodiments, ashort antisense compound targeted to SEQ ID NO: 10 is at least 95%complementary to SEQ ID NO: 10. In certain such embodiments, a shortantisense compound targeted to SEQ ID NO: 10 is 100% complementary toSEQ ID NO: 10. In certain embodiments, a short antisense compoundtargeted to SEQ ID NO: 10 includes a nucleotide sequence selected fromthe nucleotide sequences set forth in Tables 14 and 15.

Each nucleotide sequence set forth in each Tables 14 and 15 isindependent of any modification to a sugar moiety, an internucleosidelinkage, or a nucleobase. As such, short antisense compounds comprisinga nucleotide sequence as set forth in Tables 14 and 15 may comprise,independently, one or more modifications to a sugar moiety, aninternucleoside linkage, or a nucleobase. Antisense compounds describedby Isis Number (Isis NO.) indicate a combination of nucleobase sequenceand one or more modifications to a sugar moiety, an internucleosidelinkage, or a nucleobase.

Tables 14 and 15 illustrate examples of short antisense compoundstargeted to SEQ ID NO: 10. Table 14 illustrates short antisensecompounds that are 100% complementary to SEQ ID NO: 10. Table 15illustrates short antisense compounds that have one or two mismatcheswith respect to SEQ ID NO: 10. The column labeled ‘gapmer motif’indicates the wing-gap-wing motif of each short antisense compounds. Thegap segment comprises 2′-deoxynucleotides and each nucleotide of eachwing segment comprises a 2′-modified sugar. The particular 2′-modifiedsugar is also indicated in the ‘gapmer motif’ column. For example,‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten2′-deoxynucleotides is flanked by wing segments of two nucleotides,where the nucleotides of the wing segments are 2′-MOE nucleotides.Internucleoside linkages are phosphorothioate. The short antisensecompounds comprise 5-methylcytidine in place of unmodified cytosine,unless “unmodified cytosine” is listed in the gapmer motif column, inwhich case the indicated cytosines are unmodified cytosines. Forexample, “5-mC in gap only” indicates that the gap segment has5-methylcytosines, while the wing segments have unmodified cytosines.

TABLE 14 Short Antisense Compounds targeted to SEQ ID NO: 10 5′ 3′ SEQISIS Target Target Gapmer ID NO. Site Site Sequence (5′-3′) Motif NO372556 231 244 ATGAGGGTCTTCAT 2-10-2 MOE 681 372557 249 262ACCCCGGAGTAGGC 2-10-2 MOE 682 382601 249 260 CCCGGAGTAGGC 1-10-1 MOE 683372480 251 266 CAGGACCCCGGAGTAG 3-10-3 MOE 684 372481 252 267GCAGGACCCCGGAGTA 3-10-3 MOE 685 372558 252 265 AGGACCCCGGAGTA 2-10-2 MOE686 372559 253 266 CAGGACCCCGGAGT 2-10-2 MOE 687 382603 331 342CAGACCCCTCGC 1-10-1 MOE 688 382604 361 372 AGAGGATGCTGG 1-10-1 MOE 689372485 392 407 GAGCCAGGTGACAGAG 3-10-3 MOE 690 372563 393 406AGCCAGGTGACAGA 2-10-2 MOE 691 382605 397 408 TGAGCCAGGTGA 1-10-1 MOE 692372565 414 427 TTTTCCACCTTGGA 2-10-2 MOE 693 382606 482 493 CTGCAGGCCACT1-10-1 MOE 694 372497 651 666 TCACCAGCTGGATGGG 3-10-3 MOE 695 372498 652667 TTCACCAGCTGGATGG 3-10-3 MOE 696 372575 652 665 CACCAGCTGGATGG 2-10-2MOE 697 372576 653 666 TCACCAGCTGGATG 2-10-2 MOE 698 382607 655 666TCACCAGCTGGA 1-10-1 MOE 699 372499 656 671 TGTCTTCACCAGCTGG 3-10-3 MOE700 372577 657 670 GTCTTCACCAGCTG 2-10-2 MOE 701 372500 659 674GTGTGTCTTCACCAGC 3-10-3 MOE 702 372578 660 673 TGTGTCTTCACCAG 2-10-2 MOE703 372501 661 676 TTGTGTGTCTTCACCA 3-10-3 MOE 704 372579 662 675TGTGTGTCTTCACC 2-10-2 MOE 705 372502 664 679 AGGTTGTGTGTCTTCA 3-10-3 MOE706 372580 665 678 GGTTGTGTGTCTTC 2-10-2 MOE 707 372503 666 681GCAGGTTGTGTGTCTT 3-10-3 MOE 708 372581 667 680 CAGGTTGTGTGTCT 2-10-2 MOE709 372504 669 684 TCAGCAGGTTGTGTGT 3-10-3 MOE 710 372582 670 683CAGCAGGTTGTGTG 2-10-2 MOE 711 372505 671 686 GGTCAGCAGGTTGTGT 3-10-3 MOE712 372506 672 687 TGGTCAGCAGGTTGTG 3-10-3 MOE 713 372583 672 685GTCAGCAGGTTGTG 2-10-2 MOE 714 372584 673 686 GGTCAGCAGGTTGT 2-10-2 MOE715 372507 676 691 CTGGTGGTCAGCAGGT 3-10-3 MOE 716 372585 677 690TGGTGGTCAGCAGG 2-10-2 MOE 717 382608 680 691 CTGGTGGTCAGC 1-10-1 MOE 718372508 681 696 AGTTCCTGGTGGTCAG 3-10-3 MOE 719 372586 682 695GTTCCTGGTGGTCA 2-10-2 MOE 720 372509 684 699 TATAGTTCCTGGTGGT 3-10-3 MOE721 372587 685 698 ATAGTTCCTGGTGG 2-10-2 MOE 722 372510 686 701GATATAGTTCCTGGTG 3-10-3 MOE 723 372588 687 700 ATATAGTTCCTGGT 2-10-2 MOE724 372511 691 706 CCAAAGATATAGTTCC 3-10-3 MOE 725 372512 692 707TCCAAAGATATAGTTC 3-10-3 MOE 726 372589 692 705 CAAAGATATAGTTC 2-10-2 MOE727 372590 693 706 CCAAAGATATAGTT 2-10-2 MOE 728 382609 724 735CCAGGCCCATGA 1-10-1 MOE 729 372514 725 740 GGCACCCAGGCCCATG 3-10-3 MOE730 372592 726 739 GCACCCAGGCCCAT 2-10-2 MOE 731 372515 730 745CAGAAGGCACCCAGGC 3-10-3 MOE 732 372593 731 744 AGAAGGCACCCAGG 2-10-2 MOE733 382610 851 862 CCAGACATCAGG 1-10-1 MOE 734 382611 867 878GACAGGGCAGAT 1-10-1 MOE 735 382602 868 879 TGACAGGGCAGA 1-10-1 MOE 736382612 911 922 CCACTCCCATTC 1-10-1 MOE 737 372524 965 980GCCAGGCATGGAGCTC 3-10-3 MOE 738 372602 966 979 CCAGGCATGGAGCT 2-10-2 MOE739 382613 968 979 CCAGGCATGGAG 1-10-1 MOE 740 382614 987 998CAGGGTGACTGC 1-10-1 MOE 741 372525 989 1004 GTTCCGCAGGGTGACT 3-10-3 MOE742 372603 990 1003 TTCCGCAGGGTGAC 2-10-2 MOE 743 372526 992 1007GCGGTTCCGCAGGGTG 3-10-3 MOE 744 372604 993 1006 CGGTTCCGCAGGGT 2-10-2MOE 745 372530 1106 1121 TCGGCCCCAGGAGCCC 3-10-3 MOE 746 372608 11071120 CGGCCCCAGGAGCC 2-10-2 MOE 747 372531 1109 1124 CCATCGGCCCCAGGAG3-10-3 MOE 748 372609 1110 1123 CATCGGCCCCAGGA 2-10-2 MOE 749 3725321112 1127 GACCCATCGGCCCCAG 3-10-3 MOE 750 372610 1113 1126ACCCATCGGCCCCA 2-10-2 MOE 751 372533 1117 1132 TTCTGGACCCATCGGC 3-10-3MOE 752 382615 1117 1128 GGACCCATCGGC 1-10-1 MOE 753 372611 1118 1131TCTGGACCCATCGG 2-10-2 MOE 754 372536 1199 1214 CACCAGCCCCCAGGTG 3-10-3MOE 755 372614 1200 1213 ACCAGCCCCCAGGT 2-10-2 MOE 756 372537 1204 1219TAGGGCACCAGCCCCC 3-10-3 MOE 757 372615 1205 1218 AGGGCACCAGCCCC 2-10-2MOE 758 372538 1209 1224 TGGAGTAGGGCACCAG 3-10-3 MOE 759 372616 12101223 GGAGTAGGGCACCA 2-10-2 MOE 760 382616 1215 1226 CTTGGAGTAGGG 1-10-1MOE 761 372539 1218 1233 TGATGGGCTTGGAGTA 3-10-3 MOE 762 372617 12191232 GATGGGCTTGGAGT 2-10-2 MOE 763 372540 1293 1308 TGTGGTACAGGTCGAT3-10-3 MOE 764 372618 1294 1307 GTGGTACAGGTCGA 2-10-2 MOE 765 3826171294 1305 GGTACAGGTCGA 1-10-1 MOE 766 372541 1295 1310 GGTGTGGTACAGGTCG3-10-3 MOE 767 372619 1296 1309 GTGTGGTACAGGTC 2-10-2 MOE 768 3725421298 1313 CATGGTGTGGTACAGG 3-10-3 MOE 769 372620 1299 1312ATGGTGTGGTACAG 2-10-2 MOE 770 372543 1300 1315 TACATGGTGTGGTACA 3-10-3MOE 771 372621 1301 1314 ACATGGTGTGGTAC 2-10-2 MOE 772 372544 1303 1318ATGTACATGGTGTGGT 3-10-3 MOE 773 372622 1304 1317 TGTACATGGTGTGG 2-10-2MOE 774 382618 1313 1324 GCCTCCATGTAC 1-10-1 MOE 775 382619 1325 1336AGCTTCACCAGG 1-10-1 MOE 776 382620 1383 1394 GTTCACCTCCAG 1-10-1 MOE 777

TABLE 15 Short antisense compounds targeted to SEQ ID NO: 10 and having1 or 2 mismatches 5′ 3′ SEQ ISIS Target Target Gapmer ID NO Site SiteSequence (5′-3′) Motif NO 372608 151 164 CGGCCCCAGGAGCC 2-10-2 MOE 747372474 156 171 CATGCCCCAGCCGCCG 3-10-3 MOE 778 372552 157 170ATGCCCCAGCCGCC 2-10-2 MOE 779 382609 167 178 CCAGGCCCATGA 1-10-1 MOE 729372478 230 245 GATGAGGGTCTTCATG 3-10-3 MOE 780 372479 248 263GACCCCGGAGTAGGCA 3-10-3 MOE 781 382611 317 328 GACAGGGCAGAT 1-10-1 MOE735 372483 352 367 ATGCTGGAGCCAGTGC 3-10-3 MOE 782 372561 353 366TGCTGGAGCCAGTG 2-10-2 MOE 783 372562 373 386 GTCTTGGAGGGCCG 2-10-2 MOE784 382602 388 399 TGACAGGGCAGA 1-10-1 MOE 736 372613 392 405CCCAGGTGTCAGAG 2-10-2 MOE 785 372486 412 427 TTTTCCACCTTGGATC 3-10-3 MOE786 372564 413 426 TTTCCACCTTGGAT 2-10-2 MOE 787 372487 413 428TTTTTCCACCTTGGAT 3-10-3 MOE 788 372488 418 433 AGGTGTTTTTCCACCT 3-10-3MOE 789 372566 419 432 GGTGTTTTTCCACC 2-10-2 MOE 790 372489 459 474CCAGGAAGGATAGGAC 3-10-3 MOE 791 372567 460 473 CAGGAAGGATAGGA 2-10-2 MOE792 382612 475 486 CCACTCCCATTC 1-10-1 MOE 737 372490 483 498TGACACTGCAGGCCAC 3-10-3 MOE 793 372568 484 497 GACACTGCAGGCCA 2-10-2 MOE794 372491 492 507 ACATGAGGATGACACT 3-10-3 MOE 795 372569 493 506CATGAGGATGACAC 2-10-2 MOE 796 372492 503 518 GCAGAAGGTGTACATG 3-10-3 MOE797 372570 504 517 CAGAAGGTGTACAT 2-10-2 MOE 798 372493 512 527GCAGTCAGTGCAGAAG 3-10-3 MOE 799 372571 513 526 CAGTCAGTGCAGAA 2-10-2 MOE800 372496 612 627 ACACGGCCCAGTTTCG 3-10-3 MOE 801 372574 613 626CACGGCCCAGTTTC 2-10-2 MOE 802 372513 717 732 GGCCCATGATGCCATG 3-10-3 MOE803 372591 718 731 GCCCATGATGCCAT 2-10-2 MOE 804 372516 732 747TACAGAAGGCACCCAG 3-10-3 MOE 805 372594 733 746 ACAGAAGGCACCCA 2-10-2 MOE806 372518 812 827 GAAGTTGCCAGCCAAT 3-10-3 MOE 807 372596 813 826AAGTTGCCAGCCAA 2-10-2 MOE 808 372560 863 876 CAGGGCAGATCCTT 2-10-2 MOE809 372519 887 902 CAAGTAGTCTATGGTG 3-10-3 MOE 810 372597 888 901AAGTAGTCTATGGT 2-10-2 MOE 811 372520 894 909 TGGAAAGCAAGTAGTC 3-10-3 MOE812 372598 895 908 GGAAAGCAAGTAGT 2-10-2 MOE 813 372527 1013 1028GGCCAGCTTTACAAAG 3-10-3 MOE 814 372605 1014 1027 GCCAGCTTTACAAA 2-10-2MOE 815 372606 1020 1033 CGCAGGGCCAGCTT 2-10-2 MOE 816 372529 1052 1067AAAGGAATAGGTGGGA 3-10-3 MOE 817 372607 1053 1066 AAGGAATAGGTGGG 2-10-2MOE 818 372534 1144 1159 GCGAAACCAATATACT 3-10-3 MOE 819 372612 11451158 CGAAACCAATATAC 2-10-2 MOE 820 372535 1192 1207 CCCCAGGTGTCAGAGG3-10-3 MOE 821 372613 1193 1206 CCCAGGTGTCAGAG 2-10-2 MOE 822 3725451332 1347 GATTGTCAAAGAGCTT 3-10-3 MOE 823 372623 1333 1346ATTGTCAAAGAGCT 2-10-2 MOE 824 372546 1342 1357 TTGGTCTTGTGATTGT 3-10-3MOE 825 372624 1343 1356 TGGTCTTGTGATTG 2-10-2 MOE 826 372547 1352 1367AAGGCCGAATTTGGTC 3-10-3 MOE 827 372625 1353 1366 AGGCCGAATTTGGT 2-10-2MOE 828 382601 1617 1628 CCCGGAGTAGGC 1-10-1 MOE 683 382606 1971 1982CTGCAGGCCACT 1-10-1 MOE 694 382612 1988 1999 CCACTCCCATTC 1-10-1 MOE 737

In certain embodiments, a target region is nucleotides 231-267 of SEQ IDNO: 10. In certain embodiments, a short antisense compound is targetedto nucleotides 231-267 of SEQ ID NO: 10. In certain such embodiments, ashort antisense compound targeted to nucleotides 231-267 comprises anucleotide sequence selected from SEQ ID NO 681, 682, 683, 684, 685,686, or 687. In certain such embodiments, a short antisense compoundtargeted to nucleotides 231-267 of SEQ ID NO: 10 is selected from IsisNo 372556, 372557, 382601, 372480, 372481, 372558, or 372559.

In certain embodiments, a target region is nucleotides 249-267 of SEQ IDNO: 10. In certain embodiments, a short antisense compound is targetedto nucleotides 249-267 of SEQ ID NO: 10. In certain such embodiments, ashort antisense compound targeted to nucleotides 249-267 comprises anucleotide sequence selected from SEQ ID NO 683, 684, 685, 686, or 687.In certain such embodiments, a short antisense compound targeted tonucleotides 249-267 of SEQ ID NO: 10 is selected from Isis No 382601,372480, 372481, 372558, or 372559.

In certain embodiments, a target region is nucleotides 331-493 of SEQ IDNO: 10. In certain embodiments, a short antisense compound is targetedto nucleotides 331-493 of SEQ ID NO: 10. In certain such embodiments, ashort antisense compound targeted to nucleotides 331-493 comprises anucleotide sequence selected from SEQ ID NO 688, 689, 690, 691, 692,693, or 694. In certain such embodiments, a short antisense compoundtargeted to nucleotides 331-493 of SEQ ID NO: 10 is selected from IsisNo 382603, 382604, 372485, 372563, 382605, 372565, or 382606.

In certain embodiments, a target region is nucleotides 331-427 of SEQ IDNO: 10. In certain embodiments, a short antisense compound is targetedto nucleotides 331-427 of SEQ ID NO: 10. In certain such embodiments, ashort antisense compound targeted to nucleotides 331-427 comprises anucleotide sequence selected from SEQ ID NO 688, 689, 690, 691, 692, or693. In certain such embodiments, a short antisense compound targeted tonucleotides 331-427 of SEQ ID NO: 10 is selected from Isis No 382603,382604, 372485, 372563, 382605, or 372565.

In certain embodiments, a target region is nucleotides 392-408 of SEQ IDNO: 10. In certain embodiments, a short antisense compound is targetedto nucleotides 392-408 of SEQ ID NO: 10. In certain such embodiments, ashort antisense compound targeted to nucleotides 392-408 comprises anucleotide sequence selected from SEQ ID NO 690, 691, or 692. In certainsuch embodiments, a short antisense compound targeted to nucleotides392-408 of SEQ ID NO: 10 is selected from Isis No 372485, 372563, or382605.

In certain embodiments, a target region is nucleotides 651-707 of SEQ IDNO: 10. In certain embodiments, a short antisense compound is targetedto nucleotides 651-707 of SEQ ID NO: 10. In certain such embodiments, ashort antisense compound targeted to nucleotides 651-707 comprises anucleotide sequence selected from SEQ ID NO 695, 696, 697, 698, 699,700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713,714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, or728. In certain such embodiments, a short antisense compound targeted tonucleotides 651-707 of SEQ ID NO: 10 is selected from Isis No 372497,372498, 372575, 372576, 382607, 372499, 372577, 372500, 372578, 372501,372579, 372502, 372580, 372503, 372581, 372504, 372582, 372505, 372506,372583, 372584, 372507, 372585, 382608, 372508, 372586, 372509, 372587,372510, 372588, 372511, 372512, 372589, or 372590.

In certain embodiments, a target region is nucleotides 724-745 of SEQ IDNO: 10. In certain embodiments, a short antisense compound is targetedto nucleotides 724-745 of SEQ ID NO: 10. In certain such embodiments, ashort antisense compound targeted to nucleotides 724-745 comprises anucleotide sequence selected from SEQ ID NO 729, 730, 731, 732, or 733.In certain such embodiments, a short antisense compound targeted tonucleotides 724-745 of SEQ ID NO: 10 is selected from Isis No 382609,372514, 372592, 372515, or 372593.

In certain embodiments, a target region is nucleotides 651-745 of SEQ IDNO: 10. In certain embodiments, a short antisense compound is targetedto nucleotides 651-745 of SEQ ID NO: 10. In certain such embodiments, ashort antisense compound targeted to nucleotides 651-745 comprises anucleotide sequence selected from SEQ ID NO 695, 696, 697, 698, 699,700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713,714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727,728, 729, 730, 731, 732, or 733. In certain such embodiments, a shortantisense compound targeted to nucleotides 651-745 of SEQ ID NO: 10 isselected from Isis No 372497, 372498, 372575, 372576, 382607, 372499,372577, 372500, 372578, 372501, 372579, 372502, 372580, 372503, 372581,372504, 372582, 372505, 372506, 372583, 372584, 372507, 372585, 382608,372508, 372586, 372509, 372587, 372510, 372588, 372511, 372512, 372589,372590, 382609, 372514, 372592, 372515, or 372593.

In certain embodiments, a target region is nucleotides 851-922 of SEQ IDNO: 10. In certain embodiments, a short antisense compound is targetedto nucleotides 851-922 of SEQ ID NO: 10. In certain such embodiments, ashort antisense compound targeted to nucleotides 851-922 comprises anucleotide sequence selected from SEQ ID NO 734, 735, 736, or 737. Incertain such embodiments, a short antisense compound targeted tonucleotides 851-922 of SEQ ID NO: 10 is selected from Isis No 382610,382611, 382602, or 382612.

In certain embodiments, a target region is nucleotides 851-879 of SEQ IDNO: 10. In certain embodiments, a short antisense compound is targetedto nucleotides 851-879 of SEQ ID NO: 10. In certain such embodiments, ashort antisense compound targeted to nucleotides 851-879 comprises anucleotide sequence selected from SEQ ID NO 734, 735, or 736. In certainsuch embodiments, a short antisense compound targeted to nucleotides851-879 of SEQ ID NO: 10 is selected from Isis No 382610, 382611, or382602.

In certain embodiments, a target region is nucleotides 965-1007 of SEQID NO: 10. In certain embodiments, a short antisense compound istargeted to nucleotides 965-1007 of SEQ ID NO: 10. In certain suchembodiments, a short antisense compound targeted to nucleotides 965-1007comprises a nucleotide sequence selected from SEQ ID NO 738, 739, 740,741, 742, 743, 744, or 745. In certain such embodiments, a shortantisense compound targeted to nucleotides 965-1007 of SEQ ID NO: 10 isselected from Isis No 372524, 372602, 382613, 382614, 372525, 372603,372526, or 372604.

In certain embodiments, a target region is nucleotides 965-979 of SEQ IDNO: 10. In certain embodiments, a short antisense compound is targetedto nucleotides 965-979 of SEQ ID NO: 10. In certain such embodiments, ashort antisense compound targeted to nucleotides 965-979 comprises anucleotide sequence selected from SEQ ID NO 738, 739, or 740. In certainsuch embodiments, a short antisense compound targeted to nucleotides965-979 of SEQ ID NO: 10 is selected from Isis No 372524, 372602, or382613.

In certain embodiments, a target region is nucleotides 987-1007 of SEQID NO: 10. In certain embodiments, a short antisense compound istargeted to nucleotides 987-1007 of SEQ ID NO: 10. In certain suchembodiments, a short antisense compound targeted to nucleotides 987-1007comprises a nucleotide sequence selected from SEQ ID NO 741, 742, 743,744, or 745. In certain such embodiments, a short antisense compoundtargeted to nucleotides 987-1007 of SEQ ID NO: 10 is selected from IsisNo 382614, 372525, 372603, 372526, or 372604.

In certain embodiments, a target region is nucleotides 1106-1132 of SEQID NO: 10. In certain embodiments, a short antisense compound istargeted to nucleotides 1106-1132 of SEQ ID NO: 10. In certain suchembodiments, a short antisense compound targeted to nucleotides1106-1132 comprises a nucleotide sequence selected from SEQ ID NO 746,747, 748, 749, 750, 751, 752, 753, or 754. In certain such embodiments,a short antisense compound targeted to nucleotides 1106-1132 of SEQ IDNO: 10 is selected from Isis No 372530, 372608, 372531, 372609, 372532,372610, 372533, 382615, or 372611.

In certain embodiments, a target region is nucleotides 1199-1233 of SEQID NO: 10. In certain embodiments, a short antisense compound istargeted to nucleotides 1199-1233 of SEQ ID NO: 10. In certain suchembodiments, a short antisense compound targeted to nucleotides1199-1233 comprises a nucleotide sequence selected from SEQ ID NO 755,756, 757, 758, 759, 760, 761, 762, or 763. In certain such embodiments,a short antisense compound targeted to nucleotides 1199-1233 of SEQ IDNO: 10 is selected from Isis No 372536, 372614, 372537, 372615, 372538,372616, 382616, 372539, or 372617.

In certain embodiments, a target region is nucleotides 1293-1394 of SEQID NO: 10. In certain embodiments, a short antisense compound istargeted to nucleotides 1293-1394 of SEQ ID NO: 10. In certain suchembodiments, a short antisense compound targeted to nucleotides1293-1394 comprises a nucleotide sequence selected from SEQ ID NO 764,765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, or 777. Incertain such embodiments, a short antisense compound targeted tonucleotides 1293-1394 of SEQ ID NO: 10 is selected from Isis No 372540,372618, 382617, 372541, 372619, 372542, 372620, 372543, 372621, 372544,372622, 382618, 382619, or 382620.

In certain embodiments, a target region is nucleotides 1293-1336 of SEQID NO: 10. In certain embodiments, a short antisense compound istargeted to nucleotides 1293-1336 of SEQ ID NO: 10. In certain suchembodiments, a short antisense compound targeted to nucleotides1293-1336 comprises a nucleotide sequence selected from SEQ ID NO 764,765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, or 776. Incertain such embodiments, a short antisense compound targeted tonucleotides 1293-1336 of SEQ ID NO: 10 is selected from Isis No 372540,372618, 382617, 372541, 372619, 372542, 372620, 372543, 372621, 372544,372622, 382618, or 382619.

In certain embodiments, a target region is nucleotides 1293-1324 of SEQID NO: 10. In certain embodiments, a short antisense compound istargeted to nucleotides 1293-1324 of SEQ ID NO: 10. In certain suchembodiments, a short antisense compound targeted to nucleotides1293-1324 comprises a nucleotide sequence selected from SEQ ID NO 764,765, 766, 767, 768, 769, 770, 771, 772, 773, 774, or 775. In certainsuch embodiments, a short antisense compound targeted to nucleotides1293-1324 of SEQ ID NO: 10 is selected from Isis No 372540, 372618,382617, 372541, 372619, 372542, 372620, 372543, 372621, 372544, 372622,or 382618.

In certain embodiments, short antisense compounds targeted to a DGAT2nucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 nucleotides in length. In certain embodiments,short antisense compounds targeted to a DGAT2 nucleic acid are 9 to 14nucleotides in length. In certain embodiments, short antisense compoundstargeted to a DGAT2 nucleic acid are 10 to 14 nucleotides in length. Incertain embodiments, such short antisense compounds are short antisenseoligonucleotides.

In certain embodiments, short antisense compounds targeted to a DGAT2nucleic acid are short gapmers. In certain such embodiments, shortgapmers targeted to a DGAT2 nucleic acid comprise at least one highaffinity modification in one or more wings of the compound. In certainembodiments, short antisense compounds targeted to a DGAT2 nucleic acidcomprise 1 to 3 high-affinity modifications in each wing. In certainsuch embodiments, the nucleosides or nucleotides of the wing comprise a2′ modification. In certain such embodiments, the monomers of the wingare BNA's. In certain such embodiments, the monomers of the wing areselected from α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, β-D-Methyleneoxy(4′-CH₂—O-2′) BNA, Ethyleneoxy (4′-(CH₂)-2-O-2′) BNA, Aminooxy(4′-CH₂—O—N(R)-2′) BNA and Oxyamino (4′-CH₂—N(R)—O-2′) BNA. In certainembodiments, the monomers of a wing comprise a substituent at the 2′position selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H or substituted or unsubstitutedC₁-C₁₀ alkyl. In certain embodiments, the monomers of a wing are 2′MOEnucleotides.

In certain embodiments, short antisense compounds targeted to a DGAT2nucleic acid comprise a gap between the 5′ wing and the 3′ wing. Incertain embodiments the gap comprises five, six, seven, eight, nine,ten, eleven, twelve, thirteen, or fourteen monomers. In certainembodiments, the monomers of the gap are unmodifieddeoxyribonucleotides. In certain embodiments, the monomers of the gapare unmodified ribonucleotides. In certain embodiments, gapmodifications (if any) gap result in a short antisense compound that,when bound to its target nucleic acid, supports cleavage by an RNase,including, but not limited to, RNase H.

In certain embodiments, short antisense compounds targeted to a DGAT2nucleic acid have uniform monomeric linkages. In certain suchembodiments, those linkages are all phosphorothioate linkages. Incertain embodiments, the linkages are all phosphodiester linkages. Incertain embodiments, short antisense compounds targeted to a DGAT2nucleic acid have mixed backbones.

In certain embodiments, short antisense compounds targeted to a DGAT2nucleic acid are 8 monomers in length. In certain embodiments, shortantisense compounds targeted to a DGAT2 nucleic acid are 9 monomers inlength. In certain embodiments, short antisense compounds targeted to aDGAT2 nucleic acid are 10 monomers in length. In certain embodiments,short antisense compounds targeted to a DGAT2 nucleic acid are 11monomers in length. In certain embodiments, short antisense compoundstargeted to a DGAT2 nucleic acid are monomers in length. In certainembodiments, short antisense compounds targeted to a DGAT2 nucleic acidare 13 monomers in length. In certain embodiments, short antisensecompounds targeted to a DGAT2 nucleic acid are 14 monomers in length. Incertain embodiments, short antisense compounds targeted to a DGAT2nucleic acid are 15 monomers in length. In certain embodiments, shortantisense compounds targeted to a DGAT2 nucleic acid are 16 monomers inlength. In certain embodiments, short antisense compounds targeted to aDGAT2 nucleic acid comprise 9 to 15 monomers. In certain embodiments,short antisense compounds targeted to a DGAT2 nucleic acid comprise 10to 15 monomers. In certain embodiments, short antisense compoundstargeted to a DGAT2 nucleic acid comprise 12 to 14 monomers. In certainembodiments, short antisense compounds targeted to a DGAT2 nucleic acidcomprise 12 to 14 nucleotides or nucleosides.

In certain embodiments, the invention provides methods of modulatingexpression of DGAT2. In certain embodiments, such methods comprise useof one or more short antisense compound targeted to a DGAT2 nucleicacid, wherein the short antisense compound targeted to a DGAT2 nucleicacid is from about 8 to about 16, preferably 9 to 15, more preferably 9to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16linked monomers). One of ordinary skill in the art will appreciate thatthis comprehends methods of modulating expression of DGAT2 using one ormore short antisense compounds targeted to a DGAT2 nucleic acid of 8, 9,10, 11, 12, 13, 14, 15 or 16 monomers.

In certain embodiments, methods of modulating DGAT2 comprise use of ashort antisense compound targeted to a DGAT2 nucleic acid that is 8monomers in length. In certain embodiments, methods of modulating DGAT2comprise use of a short antisense compound targeted to a DGAT2 nucleicacid that is 9 monomers in length. In certain embodiments, methods ofmodulating DGAT2 comprise use of a short antisense compound targeted toa DGAT2 nucleic acid that is 10 monomers in length. In certainembodiments, methods of modulating DGAT2 comprise use of a shortantisense compound targeted to a DGAT2 nucleic acid that is 11 monomersin length. In certain embodiments, methods of modulating DGAT2 compriseuse of a short antisense compound targeted to a DGAT2 nucleic acid thatis 12 monomers in length. In certain embodiments, methods of modulatingDGAT2 comprise use of a short antisense compound targeted to a DGAT2nucleic acid that is 13 monomers in length. In certain embodiments,methods of modulating DGAT2 comprise use of a short antisense compoundtargeted to a DGAT2 nucleic acid that is 14 monomers in length. Incertain embodiments, methods of modulating DGAT2 comprise use of a shortantisense compound targeted to a DGAT2 nucleic acid that is 15 monomersin length. In certain embodiments, methods of modulating DGAT2 compriseuse of a short antisense compound targeted to a DGAT2 nucleic acid thatis 16 monomers in length.

In certain embodiments, methods of modulating expression of DGAT2comprise use of a short antisense compound targeted to a DGAT2 nucleicacid comprising 9 to 15 monomers. In certain embodiments, methods ofmodulating expression of DGAT2 comprise use of a short antisensecompound targeted to a DGAT2 nucleic acid comprising 10 to 15 monomers.In certain embodiments, methods of modulating expression of DGAT2comprise use of a short antisense compound targeted to a DGAT2 nucleicacid comprising 12 to 14 monomers. In certain embodiments, methods ofmodulating expression of DGAT2 comprise use of a short antisensecompound targeted to a DGAT2 nucleic acid comprising 12 or 14nucleotides or nucleosides.

9. PTP1B

PTP1B (also known as protein phosphatase 1B and PTPN1) is an endoplasmicreticulum (ER)-associated enzyme originally isolated as the majorprotein tyrosine phosphatase of the human placenta (Tonks et al., J.Biol. Chem., 1988, 263, 6731-6737; Tonks et al., J. Biol. Chem., 1988,263, 6722-6730).

An essential regulatory role in signaling mediated by the insulinreceptor has been established for PTP1B. In certain instances, PTP1Binteracts with and dephosphorylates the activated insulin receptor bothin vitro and in intact cells resulting in the downregulation of thesignaling pathway (Goldstein et al., Mol. Cell. Biochem., 1998, 182,91-99; Seely et al., Diabetes, 1996, 45, 1379-1385). In addition, PTP1Bmodulates the mitogenic actions of insulin (Goldstein et al., Mol. Cell.Biochem., 1998, 182, 91-99). In rat adipose cells overexpressing PTP1B,the translocation of the GLUT4 glucose transporter was inhibited,implicating PTP1B as a negative regulator of glucose transport as well(Chen et al., J. Biol. Chem., 1997, 272, 8026-8031).

Mouse knockout models lacking the PTP1B gene also point toward thenegative regulation of insulin signaling by PTP1B. Mice harboring adisrupted PTP1B gene showed increased insulin sensitivity and increasedphosphorylation of the insulin receptor. When placed on a high-fat diet,PTP1B −/− mice were resistant to weight gain and remained insulinsensitive (Elchebly et al., Science, 1999, 283, 1544-1548). Thesestudies clearly establish PTP1B as a therapeutic target in the treatmentof diabetes and obesity.

Diabetes and obesity (sometimes now collectively referred to as“diabesity”) are interrelated. Most human obesity is associated withinsulin resistance and leptin resistance. In fact obesity may have aneven greater impact on insulin action than does diabetes itself(Sindelka et al., Physiol Res., 2002, 51, 85-91). Syndrome X ormetabolic syndrome is a new term for a cluster of conditions, that, whenoccurring together, may indicate a predisposition to diabetes andcardiovascular disease. These symptoms, including high blood pressure,high triglycerides, decreased HDL and obesity, tend to appear togetherin some individuals. Because of its role in both diabetes and obesity,PTP1B is believed to be a therapeutic target for a range of metabolicconditions, including diabetes, obesity and metabolic syndrome. Byimproving blood glucose control, inhibitors of PTP1B may also be usefulin slowing, preventing, delaying or ameliorating the sequelae ofdiabetes, which include retinopathy, neuropathy, cardiovascularcomplications and nephropathy.

PTP1B, which is differentially regulated during the cell cycle(Schievella et al., Cell. Growth Differ., 1993, 4, 239-246), isexpressed in insulin sensitive tissues as two different isoforms thatarise from alternate splicing of the pre-mRNA (Shifrin and Neel, J.Biol. Chem., 1993, 268, 25376-25384). The ratio of the alternativelyspliced products is affected by growth factors, such as insulin, anddiffers in various tissues examined (Sell and Reese, Mol. Genet. Metab.,1999, 66, 189-192). In these studies the levels of the variantscorrelated with the plasma insulin concentration and percentage bodyfat. These variants may therefore be used as a biomarker for patientswith chronic hyperinsulinemia or type 2 diabetes.

Definitions

“Protein tyrosine phosphatase 1B” is the gene product or protein ofwhich expression is to be modulated by administration of a shortantisense compound. Protein tyrosine phosphatase 1B is generallyreferred to as PTP1B but may also be referred to as protein tyrosinephosphatase; PTPN1; RKPTP; protein tyrosine phosphatase, non-receptortype 1.

“PTP1B nucleic acid” means any nucleic acid encoding PTP1B. For example,in certain embodiments, a PTP1B nucleic acid includes, withoutlimitation, a DNA sequence encoding PTP1B, an RNA sequence transcribedfrom DNA encoding PTP1B, and an mRNA sequence encoding PTP1B.

“PTP1B mRNA” means an mRNA encoding a PTP1B protein.

Therapeutic Indications

Antisense technology is an effective means for reducing PTP1B expressionand has proven to be uniquely useful in a number of therapeutic,diagnostic, and research applications. As such, in certain embodiments,the present invention provides compounds targeted to a nucleic acidencoding PTP1B, which modulate the expression of PTP1B. Further providedherein are short antisense compounds capable of effectively inhibitingPTP1B expression.

In certain therapeutics, a subject, suspected of having a disease ordisorder which can be treated by modulating the expression of PTP1B istreated by administering one or more short antisense compounds targetedto a nucleic acid encoding PTP1B. For example, in one non-limitingembodiment, the methods comprise the step of administering to an animala therapeutically effective amount of a short antisense compound. Theshort antisense compounds of the present invention effectively inhibitthe activity of PTP1B or inhibit the expression of PTP1B. In oneembodiment, the activity or expression of PTP1B in a subject isinhibited by at least 10%, by at least 20%, by at least 25%, by at least30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%,by at least 75%, by at least 80%, by at least 85%, by at least 90%, byat least 95%, by at least 98%, by at least 99%, or by 100%. In certainembodiments, activity or expression of PTP1B in a subject is inhibitedby about 30%. In certain embodiments, the activity or expression ofPTP1B in a subject is inhibited by 50% or more.

The reduction of the expression of PTP1B may be measured, for example,in blood, plasma, serum, adipose tissue, liver or any other body fluid,tissue or organ of the animal. Preferably, the cells contained withinsaid fluids, tissues or organs being analyzed contain a nucleic acidmolecule encoding PTP1B and/or the PTP1B protein itself.

Certain pharmaceutical and other compositions comprising the compoundsof the invention are also provided. In certain embodiments shortantisense compounds targeted to a PTP1B nucleic acid are utilized inpharmaceutical compositions by adding an effective amount of a compoundto a suitable pharmaceutically acceptable diluent or carrier.

The short antisense compounds targeting PTP1B may have any one or moreproperties or characteristics of the short antisense compounds generallydescribed herein. In certain embodiments, short antisense compoundstargeting a PTP1B nucleic acid have a motif (wing-deoxy gap-wing)selected from 1-12-1, 1-1-10-2, 2-10-1-1, 3-10-3, 2-10-3, 2-10-2,1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or 1-6-1, more preferably1-10-1, 2-10-2, 3-10-3, and 1-9-2.

In certain embodiments provided herein are methods of treating anindividual by administering one or more short antisense compoundtargeted to a PTP1B nucleic acid or a pharmaceutical compositioncomprising such compound. Further provided are methods of treating asubject having a disease or conditions associated with PTP1B activity byadministering a short antisense compound targeted to a PTP1B nucleicacid. Diseases and conditions associated with PTP1B include but are notlimited to high blood glucose or hyperglycemia, prediabetes, diabetes,Type 2 diabetes, metabolic syndrome, obesity and insulin resistance.Therefore, provided herein are methods of treating to high blood glucoseor hyperglycemia, prediabetes, diabetes, Type 2 diabetes, metabolicsyndrome, obesity and insulin resistance by administering a shortantisense compound targeted to a PTP1B nucleic acid.

In certain embodiments the present invention provides compositions andmethods for decreasing blood glucose levels in a subject or forpreventing or delaying the onset of a rise in blood glucose levels in asubject, by administering to the subject a short antisense inhibitor ofPTP1B expression.

In certain embodiments, the present invention provides compositions andmethods for improving insulin sensitivity in a subject or for preventingor delaying the onset of insulin resistance in a subject, byadministering to the subject a short antisense inhibitor of PTP1Bexpression.

In certain embodiments, the present invention provides compositions andmethods for treating a metabolic condition in a subject or forpreventing or delaying the onset of a metabolic condition in a subject,by administering to the subject a short antisense compound targeted to aPTP1B nucleic acid. Such metabolic condition may be any metaboliccondition associated with PTP1B expression, including but not limited todiabetes and obesity. Also provided are methods of reducing adiposity.Also provided is a method of treating obesity wherein metabolic rate isincreased.

In certain embodiments, the subject has Type 2 diabetes. In certainembodiments the subject exhibits elevated HbA1c levels In certainembodiments, HbA1c levels are at least about 6%, at least about 7%, atleast about 8%, at least about 9%, at least about 10% or at least about11%. In preferred embodiments, HbA_(1c) levels are reduced to about 7%or below about 7%. In certain embodiments, the subject exhibits anelevated body mass index In certain embodiments, the elevated body massindex is greater than 25 kg/m2. In certain embodiments, the subjectexhibits hyperglycemia or elevated blood glucose levels. In a particularembodiment, the blood glucose levels are fasting blood glucose levels.In certain embodiments, the elevated fasting blood glucose levels are atleast 130 mg/dL. In certain embodiments, the subject exhibitshyperglycemia prior to the start of treatment or exhibits fasting bloodglucose levels above about 130 mg/dL, baseline HbA_(1c) levels of atleast about 7%, or body mass index of greater than 25 kg/m² or anycombination thereof.

In certain embodiments a method of reducing one or more such levels byadministering a short antisense compound targeted to a PTP1B nucleicacid is provided. For example, provided is a method of reducing fastingglucose levels, HbA_(1c) levels or, body mass index levels or anycombination thereof in a subject by administering to a subject a shortantisense compound targeting PTP1B. Fasting glucose may be fasting bloodglucose, fasting serum glucose, or fasting plasma glucose. In someembodiments, fasting plasma glucose levels are reduced by at least about25 mg/dL or by at least about 10 mg/dL. In a certain embodiments, saidsubject does not achieve normal glucose levels on a therapeutic regimenof a glucose-lowering agent such as insulin, sulfonylurea, or metformin.

In certain embodiments the invention provides methods of altering lipidlevels. Certain such methods reduce cholesterol, LDL and/or VLDL levelsor any combination thereof in a subject by administering to the subjecta short antisense compound targeted to a PTP1B nucleic acid. In certainembodiments HDL levels in a subject are increased by administering tothe subject a short antisense compound targeted to a PTP1B nucleic acid.In certain embodiments, LDL:HDL ratio and/or total cholesterol:HDL ratioin a subject is reduced by administering to the subject a shortantisense compound targeted to a PTP1B nucleic acid. In certainembodiments HDL:LDL ratio and/or HDL:total cholesterol ratio in asubject's increased by administering to the subject a short antisensecompound targeted to a PTP1B nucleic acid. In certain embodiments lipidprofile in a subject is improved by increasing HDL, lowering LDL,lowering VLDL, lowering triglycerides, lowering apolipoprotein B levels,or lowering total cholesterol levels, or a combination thereof, byadministering to the subject a short antisense compound targeted to aPTP1B nucleic acid. In such embodiments, the subject is an animal,including a human.

Combination Therapy

In certain embodiments, one or more pharmaceutical compositionscomprising a short antisense compound targeted to a PTP1B nucleic acidare co-administered with one or more other pharmaceutical agents. Incertain embodiments, such one or more other pharmaceutical agents aredesigned to treat the same disease or condition as the one or morepharmaceutical compositions of the present invention. In certainembodiments, such one or more other pharmaceutical agents are designedto treat a different disease or condition as the one or morepharmaceutical compositions of the present invention. In certainembodiments, such one or more other pharmaceutical agents are designedto treat an undesired effect of one or more pharmaceutical compositionsof the present invention. In certain embodiments, one or morepharmaceutical compositions of the present invention are co-administeredwith another pharmaceutical agent to treat an undesired effect of thatother pharmaceutical agent. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are administered at the same time. Incertain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areadministered at different times. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are prepared together in a singleformulation. In certain embodiments, one or more pharmaceuticalcompositions of the present invention and one or more otherpharmaceutical agents are prepared separately.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition comprising a shortantisense compound targeted to a PTP1B nucleic acid includeglucose-lowering agents and therapies. In some embodiments, theglucose-lowering agent is a PPAR agonist (gamma, dual, or pan), adipeptidyl peptidase (IV) inhibitor, a GLP-1 analog, insulin or aninsulin analog, an insulin secretagogue, a SGLT2 inhibitor, a humanamylin analog, a biguanide, an alpha-glucosidase inhibitor, ameglitinide, a thiazolidinedione, or a sulfonylurea.

In some embodiments, the glucose-lowering therapeutic is a GLP-1 analog.In some embodiments, the GLP-1 analog is exendin-4 or liraglutide.

In other embodiments, the glucose-lowering therapeutic is asulfonylurea. In some embodiments, the sulfonylurea is acetohexamide,chlorpropamide, tolbutamide, tolazamide, glimepiride, a glipizide, aglyburide, or a gliclazide.

In some embodiments, the glucose lowering drug is a biguanide. In someembodiments, the biguanide is metformin, and in some embodiments, bloodglucose levels are decreased without increased lactic acidosis ascompared to the lactic acidosis observed after treatment with metforminalone.

In some embodiments, the glucose lowering drug is a meglitinide. In someembodiments, the meglitinide is nateglinide or repaglinide.

In some embodiments, the glucose-lowering drug is a thiazolidinedione.In some embodiments, the thiazolidinedione is pioglitazone,rosiglitazone, or troglitazone. In some embodiments, blood glucoselevels are decreased without greater weight gain than observed withrosiglitazone treatment alone.

In some embodiments, the glucose-lowering drug is an alpha-glucosidaseinhibitor. In some embodiments, the alpha-glucosidase inhibitor isacarbose or miglitol.

In a certain embodiment, a co-administered glucose-lowering agent isISIS 113715.

In a certain embodiment, glucose-lowering therapy is therapeuticlifestyle change.

In certain such embodiments, the glucose-lowering agent is administeredprior to administration of a pharmaceutical composition of the presentinvention. In certain such embodiments, the glucose-lowering agent isadministered following administration of a pharmaceutical composition ofthe present invention. In certain such embodiments the glucose-loweringagent is administered at the same time as a pharmaceutical compositionof the present invention. In certain such embodiments the dose of aco-administered glucose-lowering agent is the same as the dose thatwould be administered if the glucose-lowering agent was administeredalone. In certain such embodiments the dose of a co-administeredglucose-lowering agent is lower than the dose that would be administeredif the glucose-lowering agent was administered alone. In certain suchembodiments the dose of a co-administered glucose-lowering agent isgreater than the dose that would be administered if the glucose-loweringagent was administered alone.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition comprising a shortantisense compound targeted to a PTP1B nucleic acid includelipid-lowering agents. Such lipid lowering agents are discussedelsewhere in the application and are included here with respect toPTP1B. Such lipid lowering agents may be administered as described abovefor glucose lowering agents.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition comprising a shortantisense compound targeted to a PTP1B nucleic acid include anti-obesityagents therapeutics. Such anti-obesity agents therapeutics may beadministered as described above for glucose lowering agents.

Further provided is a method of administering a short antisense compoundtargeted to a PTP1B nucleic acid via injection and further includingadministering a topical steroid at the injection site.

Medicaments

Also provided herein are uses of a short antisense compound which istargeted to a PTP1B nucleic acid for the preparation of a medicament forreducing blood glucose levels including fasting glucose levels, andHbA_(1c) levels, body mass index levels or any combination thereof. Themedicament can be administered during a loading period and a maintenanceperiod. In some embodiments, the medicament is administeredsubcutaneously or intravenously. In other embodiments, theadministration of said medicament occurs at least once daily, at leastonce weekly, or at least once monthly. In a particular embodiment theshort antisense compound present in the medicament is administered in adose lower than a short antisense compound with a longer sequence andparticularly a sequence 20 or more nucleobases. The medicament may beadministered to a subject that exhibits high blood glucose orhyperglycemia, prediabetes, diabetes, Type 2 diabetes, metabolicsyndrome, obesity and insulin resistance.

Other aspects and advantages of short antisense compounds are providedherein. All aspect and advantages disclosed herein and specifically withregard to other targets is applicable with regard to compositionsincluding short antisense compounds targeted to a PTP1B nucleic acid andmethods of their use.

Certain Short Antisense Compounds Targeted to a PTP1B Nucleic Acid

In certain embodiments, short antisense compounds are targeted to aPTP1B nucleic acid having the sequence of GENBANK® Accession No.NM_(—)002827.2, incorporated herein as SEQ ID NO: 11 or the nucleotides14178000 to 1425600 of the sequence of GENBANK® Accession No.NT_(—)011362.9, incorporated herein as SEQ ID NO: 12. In certain suchembodiments, a short antisense compound targeted to SEQ ID NO: 11 is atleast 90% complementary to SEQ ID NO: 1. In certain such embodiments, ashort antisense compound targeted to SEQ ID NO: 11 is at least 95%complementary to SEQ ID NO:11. In certain such embodiments, a shortantisense compound targeted to SEQ ID NO: 12 is 100% complementary toSEQ ID NO: 12. In certain such embodiments, a short antisense compoundtargeted to SEQ ID NO: 12 is at least 90% complementary to SEQ ID NO:12. In certain such embodiments, a short antisense compound targeted toSEQ ID NO: 12 is at least 95% complementary to SEQ ID NO: 12. In certainsuch embodiments, a short antisense compound targeted to SEQ ID NO: 12is 100% complementary to SEQ ID NO: 12.

In certain embodiments, a short antisense compound targeted to SEQ IDNO: 11 comprises a nucleotide sequence selected from the nucleotidesequences set forth in Tables 16 and 17. In certain embodiments, a shortantisense compound targeted to SEQ ID NO: 12 comprises a nucleotidesequence selected from the nucleotide sequences set forth in Tables 18and 19.

Each nucleotide sequence set forth in each Tables 16, 17, 18, and 19 isindependent of any modification to a sugar moiety, an internucleosidelinkage, or a nucleobase. As such, short antisense compounds comprisinga nucleotide sequence as set forth in Tables 16, 17, 18, and 19 maycomprise, independently, one or more modifications to a sugar moiety, aninternucleoside linkage, or a nucleobase. Antisense compounds describedby Isis Number (Isis NO.) indicate a combination of nucleobase sequenceand one or more modifications to a sugar moiety, an internucleosidelinkage, or a nucleobase.

Tables 16 and 17 illustrate examples of short antisense compoundstargeted to SEQ ID NO: 11. Table 16 illustrates short antisensecompounds that are 100% complementary to SEQ ID NO: 11. Table 17illustrates short antisense compounds that have one or two mismatcheswith respect to SEQ ID NO: 11. Table 18 illustrates short antisensecompounds that are 100% complementary to SEQ ID NO: 12. Table 19illustrates short antisense compounds that have 1 or 2 mismatches withrespect to SEQ ID NO: 12. The column labeled ‘gapmer motif’ indicatesthe wing-gap-wing motif of each short antisense compounds. The gapsegment comprises 2′-deoxynucleotides and each nucleotide of each wingsegment comprises a 2′-modified sugar. The particular 2′-modified sugaris also indicated in the ‘gapmer motif’ column. For example, ‘2-10-2MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten2′-deoxynucleotides is flanked by wing segments of two nucleotides,where the nucleotides of the wing segments are 2′-MOE nucleotides.Internucleoside linkages are phosphorothioate. The short antisensecompounds comprise 5-methylcytidine in place of unmodified cytosine,unless “unmodified cytosine” is listed in the gapmer motif column, inwhich case the indicated cytosines are unmodified cytosines. Forexample, “5-mC in gap only” indicates that the gap segment has5-methylcytosines, while the wing segments have unmodified cytosines.

TABLE 16 Short Antisense Compounds targeted to SEQ ID NO: 11 5′ 3′ SEQISIS Target Target Gapmer ID NO. Site Site Sequence (5′-3′) Motif NO147022 177 188 TTGTCGATCTCC 1-10-1 MOE 886 147023 178 189 CTTGTCGATCTC1-10-1 MOE 859 147024 179 190 CCTTGTCGATCT 1-10-1 MOE 853 147019 195 206TCGATCTCCTCG 1-10-1 MOE 877 147020 196 207 GTCGATCTCCTC 1-10-1 MOE 868147021 197 208 TGTCGATCTCCT 1-10-1 MOE 882 147022 198 209 TTGTCGATCTCC1-10-1 MOE 886 147023 199 210 CTTGTCGATCTC 1-10-1 MOE 859 147024 200 211CCTTGTCGATCT 1-10-1 MOE 853 147025 201 212 GCCTTGTCGATC 1-10-1 MOE 865147026 202 213 AGCCTTGTCGAT 1-10-1 MOE 835 147027 203 214 CAGCCTTGTCGA1-10-1 MOE 843 147028 204 215 CCAGCCTTGTCG 1-10-1 MOE 846 147073 204 215CACTGATCCTGC 1-10-1 MOE 842 147029 205 216 CCCAGCCTTGTC 1-10-1 MOE 848147030 206 217 TCCCAGCCTTGT 1-10-1 MOE 874 147036 212 223 CCCAGTTCCCAG1-10-1 MOE 849 147037 213 224 GCCCAGTTCCCA 1-10-1 MOE 863 147038 214 225CGCCCAGTTCCC 1-10-1 MOE 855 147039 215 226 CCGCCCAGTTCC 1-10-1 MOE 850147040 216 227 GCCGCCCAGTTC 1-10-1 MOE 864 147041 217 228 AGCCGCCCAGTT1-10-1 MOE 834 147073 311 322 CACTGATCCTGC 1-10-1 MOE 842 147042 323 334GGTCAAAAGGGC 1-10-1 MOE 866 147043 324 335 TGGTCAAAAGGG 1-10-1 MOE 881147044 325 336 GTGGTCAAAAGG 1-10-1 MOE 869 147045 326 337 TGTGGTCAAAAG1-10-1 MOE 883 147046 327 338 CTGTGGTCAAAA 1-10-1 MOE 858 147047 328 339ACTGTGGTCAAA 1-10-1 MOE 833 147051 332 343 TCCGACTGTGGT 1-10-1 MOE 875147052 333 344 ATCCGACTGTGG 1-10-1 MOE 837 147053 334 345 AATCCGACTGTG1-10-1 MOE 829 147054 335 346 TAATCCGACTGT 1-10-1 MOE 871 147055 336 347TTAATCCGACTG 1-10-1 MOE 884 147056 337 348 TTTAATCCGACT 1-10-1 MOE 887147057 338 349 ATTTAATCCGAC 1-10-1 MOE 839 147058 339 350 AATTTAATCCGA1-10-1 MOE 830 147059 340 351 CAATTTAATCCG 1-10-1 MOE 840 147060 341 352GCAATTTAATCC 1-10-1 MOE 861 147061 342 353 TGCAATTTAATC 1-10-1 MOE 879147045 679 690 TGTGGTCAAAAG 1-10-1 MOE 883 147046 680 691 CTGTGGTCAAAA1-10-1 MOE 858 147045 787 798 TGTGGTCAAAAG 1-10-1 MOE 883 147046 788 799CTGTGGTCAAAA 1-10-1 MOE 858 147066 816 827 CCTGCACTGACG 1-10-1 MOE 851404131 992 1005 ACCTTCGATCACAG 2-10-2 MOE 831 147062 1024 1035CACTGACGAGTC 1-10-1 MOE 841 147063 1025 1036 GCACTGACGAGT 1-10-1 MOE 862147064 1026 1037 TGCACTGACGAG 1-10-1 MOE 880 147065 1027 1038CTGCACTGACGA 1-10-1 MOE 857 147066 1028 1039 CCTGCACTGACG 1-10-1 MOE 851147067 1029 1040 TCCTGCACTGAC 1-10-1 MOE 876 147068 1030 1041ATCCTGCACTGA 1-10-1 MOE 838 147069 1031 1042 GATCCTGCACTG 1-10-1 MOE 860147070 1032 1043 TGATCCTGCACT 1-10-1 MOE 878 147071 1033 1044CTGATCCTGCAC 1-10-1 MOE 856 147072 1034 1045 ACTGATCCTGCA 1-10-1 MOE 832147073 1035 1046 CACTGATCCTGC 1-10-1 MOE 842 147067 1199 1210TCCTGCACTGAC 1-10-1 MOE 876 147040 1288 1299 GCCGCCCAGTTC 1-10-1 MOE 864147040 1396 1407 GCCGCCCAGTTC 1-10-1 MOE 864 147022 1868 1879TTGTCGATCTCC 1-10-1 MOE 886 147023 1869 1880 CTTGTCGATCTC 1-10-1 MOE 859147024 1870 1881 CCTTGTCGATCT 1-10-1 MOE 853 147019 1886 1897TCGATCTCCTCG 1-10-1 MOE 877 147020 1887 1898 GTCGATCTCCTC 1-10-1 MOE 868147021 1888 1899 TGTCGATCTCCT 1-10-1 MOE 882 147022 1889 1900TTGTCGATCTCC 1-10-1 MOE 886 147023 1890 1901 CTTGTCGATCTC 1-10-1 MOE 859147025 1892 1903 GCCTTGTCGATC 1-10-1 MOE 865 147027 1894 1905CAGCCTTGTCGA 1-10-1 MOE 843 147028 1895 1906 CCAGCCTTGTCG 1-10-1 MOE 846147030 1897 1908 TCCCAGCCTTGT 1-10-1 MOE 874 147037 1904 1915GCCCAGTTCCCA 1-10-1 MOE 863 147038 1905 1916 CGCCCAGTTCCC 1-10-1 MOE 855147040 1907 1918 GCCGCCCAGTTC 1-10-1 MOE 864 147041 1908 1919AGCCGCCCAGTT 1-10-1 MOE 834 147022 1976 1987 TTGTCGATCTCC 1-10-1 MOE 886147023 1977 1988 CTTGTCGATCTC 1-10-1 MOE 859 147024 1978 1989CCTTGTCGATCT 1-10-1 MOE 853 147020 1995 2006 GTCGATCTCCTC 1-10-1 MOE 868147021 1996 2007 TGTCGATCTCCT 1-10-1 MOE 882 147022 1997 2008TTGTCGATCTCC 1-10-1 MOE 886 147023 1998 2009 CTTGTCGATCTC 1-10-1 MOE 859147024 1999 2010 CCTTGTCGATCT 1-10-1 MOE 853 147025 2000 2011GCCTTGTCGATC 1-10-1 MOE 865 147026 2001 2012 AGCCTTGTCGAT 1-10-1 MOE 835147027 2002 2013 CAGCCTTGTCGA 1-10-1 MOE 843 147028 2003 2014CCAGCCTTGTCG 1-10-1 MOE 846 147029 2004 2015 CCCAGCCTTGTC 1-10-1 MOE 848147030 2005 2016 TCCCAGCCTTGT 1-10-1 MOE 874 147036 2011 2022CCCAGTTCCCAG 1-10-1 MOE 849 147037 2012 2023 GCCCAGTTCCCA 1-10-1 MOE 863147038 2013 2024 CGCCCAGTTCCC 1-10-1 MOE 855 147039 2014 2025CCGCCCAGTTCC 1-10-1 MOE 850 147040 2015 2026 GCCGCCCAGTTC 1-10-1 MOE 864147041 2016 2027 AGCCGCCCAGTT 1-10-1 MOE 834 404199 2366 2379GGTCATGCACAGGC 2-10-2 MOE 867 404134 2369 2382 TCAGGTCATGCACA 2-10-2 MOE873 404132 2548 2561 CCTTGGAATGTCTG 2-10-2 MOE 852 147020 2613 2624GTCGATCTCCTC 1-10-1 MOE 868 147020 2721 2732 GTCGATCTCCTC 1-10-1 MOE 868404133 3289 3302 TATTCCATGGCCAT 2-10-2 MOE 872 147032 6220 6231GTTCCCAGCCTT 1-10-1 MOE 870 147033 6221 6232 AGTTCCCAGCCT 1-10-1 MOE 836147034 6222 6233 CAGTTCCCAGCC 1-10-1 MOE 844 147044 6288 6299GTGGTCAAAAGG 1-10-1 MOE 869 147045 6289 6300 TGTGGTCAAAAG 1-10-1 MOE 883147032 6329 6340 GTTCCCAGCCTT 1-10-1 MOE 870 147033 6330 6341AGTTCCCAGCCT 1-10-1 MOE 836 147034 6331 6342 CAGTTCCCAGCC 1-10-1 MOE 844147044 6397 6408 GTGGTCAAAAGG 1-10-1 MOE 869 147045 6398 6409TGTGGTCAAAAG 1-10-1 MOE 883 147058 7057 7068 AATTTAATCCGA 1-10-1 MOE 830147059 7058 7069 CAATTTAATCCG 1-10-1 MOE 840 147060 7059 7070GCAATTTAATCC 1-10-1 MOE 861 147058 7166 7177 AATTTAATCCGA 1-10-1 MOE 830147059 7167 7178 CAATTTAATCCG 1-10-1 MOE 840 147041 8084 8095AGCCGCCCAGTT 1-10-1 MOE 834 147041 8192 8203 AGCCGCCCAGTT 1-10-1 MOE 834147027 8630 8641 CAGCCTTGTCGA 1-10-1 MOE 843 147028 8631 8642CCAGCCTTGTCG 1-10-1 MOE 846 147027 8738 8749 CAGCCTTGTCGA 1-10-1 MOE 843147028 8739 8750 CCAGCCTTGTCG 1-10-1 MOE 846 147043 10957 10968TGGTCAAAAGGG 1-10-1 MOE 881 147044 10958 10969 GTGGTCAAAAGG 1-10-1 MOE869 147043 11065 11076 TGGTCAAAAGGG 1-10-1 MOE 881 147044 11066 11077GTGGTCAAAAGG 1-10-1 MOE 869 147071 11605 11616 CTGATCCTGCAC 1-10-1 MOE856 147070 11611 11622 TGATCCTGCACT 1-10-1 MOE 878 147071 11612 11623CTGATCCTGCAC 1-10-1 MOE 856 147072 12294 12305 ACTGATCCTGCA 1-10-1 MOE832 147072 12299 12310 ACTGATCCTGCA 1-10-1 MOE 832 147030 12805 12816TCCCAGCCTTGT 1-10-1 MOE 874 147031 12806 12817 TTCCCAGCCTTG 1-10-1 MOE885 147053 12939 12950 AATCCGACTGTG 1-10-1 MOE 829 147030 12986 12997TCCCAGCCTTGT 1-10-1 MOE 874 147031 12987 12998 TTCCCAGCCTTG 1-10-1 MOE885 147053 13120 13131 AATCCGACTGTG 1-10-1 MOE 829 147051 13162 13173TCCGACTGTGGT 1-10-1 MOE 875 147061 13316 13327 TGCAATTTAATC 1-10-1 MOE879 147047 13339 13350 ACTGTGGTCAAA 1-10-1 MOE 833 147029 14058 14069CCCAGCCTTGTC 1-10-1 MOE 848 147029 14239 14250 CCCAGCCTTGTC 1-10-1 MOE848 147067 15560 15571 TCCTGCACTGAC 1-10-1 MOE 876 147068 15561 15572ATCCTGCACTGA 1-10-1 MOE 838 147067 15742 15753 TCCTGCACTGAC 1-10-1 MOE876 147069 15744 15755 GATCCTGCACTG 1-10-1 MOE 860 147042 16561 16572GGTCAAAAGGGC 1-10-1 MOE 866 147042 16727 16738 GGTCAAAAGGGC 1-10-1 MOE866 147030 17619 17630 TCCCAGCCTTGT 1-10-1 MOE 874 147064 17762 17773TGCACTGACGAG 1-10-1 MOE 880 147030 17787 17798 TCCCAGCCTTGT 1-10-1 MOE874 147064 17930 17941 TGCACTGACGAG 1-10-1 MOE 880 147042 19201 19212GGTCAAAAGGGC 1-10-1 MOE 866 147042 19369 19380 GGTCAAAAGGGC 1-10-1 MOE866 147027 21190 21201 CAGCCTTGTCGA 1-10-1 MOE 843 147028 21191 21202CCAGCCTTGTCG 1-10-1 MOE 846 147027 21358 21369 CAGCCTTGTCGA 1-10-1 MOE843 147028 21359 21370 CCAGCCTTGTCG 1-10-1 MOE 846 147070 22021 22032TGATCCTGCACT 1-10-1 MOE 878 147070 22189 22200 TGATCCTGCACT 1-10-1 MOE878 147047 22606 22617 ACTGTGGTCAAA 1-10-1 MOE 833 147043 24318 24329TGGTCAAAAGGG 1-10-1 MOE 881 147044 24319 24330 GTGGTCAAAAGG 1-10-1 MOE869 147045 24320 24331 TGTGGTCAAAAG 1-10-1 MOE 883 147046 24321 24332CTGTGGTCAAAA 1-10-1 MOE 858 147043 24486 24497 TGGTCAAAAGGG 1-10-1 MOE881 147044 24487 24498 GTGGTCAAAAGG 1-10-1 MOE 869 147046 24489 24500CTGTGGTCAAAA 1-10-1 MOE 858 147047 24490 24501 ACTGTGGTCAAA 1-10-1 MOE833 147040 25065 25076 GCCGCCCAGTTC 1-10-1 MOE 864 147041 25066 25077AGCCGCCCAGTT 1-10-1 MOE 834 147046 25160 25171 CTGTGGTCAAAA 1-10-1 MOE858 147039 25232 25243 CCGCCCAGTTCC 1-10-1 MOE 850 147040 25233 25244GCCGCCCAGTTC 1-10-1 MOE 864 147041 25234 25245 AGCCGCCCAGTT 1-10-1 MOE834 147046 25328 25339 CTGTGGTCAAAA 1-10-1 MOE 858 147057 25508 25519ATTTAATCCGAC 1-10-1 MOE 839 147061 25512 25523 TGCAATTTAATC 1-10-1 MOE879 147057 25676 25687 ATTTAATCCGAC 1-10-1 MOE 839 147069 28878 28889GATCCTGCACTG 1-10-1 MOE 860 147070 28879 28890 TGATCCTGCACT 1-10-1 MOE878 147053 30133 30144 AATCCGACTGTG 1-10-1 MOE 829 147053 30278 30289AATCCGACTGTG 1-10-1 MOE 829 147054 30864 30875 TAATCCGACTGT 1-10-1 MOE871 147043 30985 30996 TGGTCAAAAGGG 1-10-1 MOE 881 147054 31011 31022TAATCCGACTGT 1-10-1 MOE 871 147043 31133 31144 TGGTCAAAAGGG 1-10-1 MOE881 147036 32233 32244 CCCAGTTCCCAG 1-10-1 MOE 849 147072 32372 32383ACTGATCCTGCA 1-10-1 MOE 832 147072 32520 32531 ACTGATCCTGCA 1-10-1 MOE832 147069 33056 33067 GATCCTGCACTG 1-10-1 MOE 860 147070 33057 33068TGATCCTGCACT 1-10-1 MOE 878 147071 33058 33069 CTGATCCTGCAC 1-10-1 MOE856 147051 33126 33137 TCCGACTGTGGT 1-10-1 MOE 875 147070 33205 33216TGATCCTGCACT 1-10-1 MOE 878 147071 33206 33217 CTGATCCTGCAC 1-10-1 MOE856 147051 33274 33285 TCCGACTGTGGT 1-10-1 MOE 875 147046 33318 33329CTGTGGTCAAAA 1-10-1 MOE 858 147049 33321 33332 CGACTGTGGTCA 1-10-1 MOE854 147051 33323 33334 TCCGACTGTGGT 1-10-1 MOE 875 147046 33466 33477CTGTGGTCAAAA 1-10-1 MOE 858 147047 33467 33478 ACTGTGGTCAAA 1-10-1 MOE833 147051 33471 33482 TCCGACTGTGGT 1-10-1 MOE 875 147046 33640 33651CTGTGGTCAAAA 1-10-1 MOE 858 147051 33645 33656 TCCGACTGTGGT 1-10-1 MOE875 147046 33788 33799 CTGTGGTCAAAA 1-10-1 MOE 858 147051 33793 33804TCCGACTGTGGT 1-10-1 MOE 875 147059 35437 35448 CAATTTAATCCG 1-10-1 MOE840 147060 35438 35449 GCAATTTAATCC 1-10-1 MOE 861 147060 35586 35597GCAATTTAATCC 1-10-1 MOE 861 147021 36093 36104 TGTCGATCTCCT 1-10-1 MOE882 147061 36250 36261 TGCAATTTAATC 1-10-1 MOE 879 147061 36398 36409TGCAATTTAATC 1-10-1 MOE 879 147073 37485 37496 CACTGATCCTGC 1-10-1 MOE842 147073 37633 37644 CACTGATCCTGC 1-10-1 MOE 842 147043 40214 40225TGGTCAAAAGGG 1-10-1 MOE 881 147061 40353 40364 TGCAATTTAATC 1-10-1 MOE879 147043 40362 40373 TGGTCAAAAGGG 1-10-1 MOE 881 147061 40501 40512TGCAATTTAATC 1-10-1 MOE 879 147031 42527 42538 TTCCCAGCCTTG 1-10-1 MOE885 147032 42528 42539 GTTCCCAGCCTT 1-10-1 MOE 870 147034 42530 42541CAGTTCCCAGCC 1-10-1 MOE 844 147031 42675 42686 TTCCCAGCCTTG 1-10-1 MOE885 147032 42676 42687 GTTCCCAGCCTT 1-10-1 MOE 870 147033 42677 42688AGTTCCCAGCCT 1-10-1 MOE 836 147034 42678 42689 CAGTTCCCAGCC 1-10-1 MOE844 147074 43848 43859 CCACTGATCCTG 1-10-1 MOE 845 147074 43996 44007CCACTGATCCTG 1-10-1 MOE 845 147051 45402 45413 TCCGACTGTGGT 1-10-1 MOE875 147051 45550 45561 TCCGACTGTGGT 1-10-1 MOE 875 147074 46125 46136CCACTGATCCTG 1-10-1 MOE 845 147057 46313 46324 ATTTAATCCGAC 1-10-1 MOE839 147058 46314 46325 AATTTAATCCGA 1-10-1 MOE 830 147059 46315 46326CAATTTAATCCG 1-10-1 MOE 840 147061 46317 46328 TGCAATTTAATC 1-10-1 MOE879 147057 46461 46472 ATTTAATCCGAC 1-10-1 MOE 839 147059 46463 46474CAATTTAATCCG 1-10-1 MOE 840 147061 46465 46476 TGCAATTTAATC 1-10-1 MOE879 147058 47413 47424 AATTTAATCCGA 1-10-1 MOE 830 147073 48221 48232CACTGATCCTGC 1-10-1 MOE 842 147073 48369 48380 CACTGATCCTGC 1-10-1 MOE842 147074 48370 48381 CCACTGATCCTG 1-10-1 MOE 845 147027 48566 48577CAGCCTTGTCGA 1-10-1 MOE 843 147027 48714 48725 CAGCCTTGTCGA 1-10-1 MOE843 147028 48715 48726 CCAGCCTTGTCG 1-10-1 MOE 846 147067 49050 49061TCCTGCACTGAC 1-10-1 MOE 876 147068 49051 49062 ATCCTGCACTGA 1-10-1 MOE838 147067 49198 49209 TCCTGCACTGAC 1-10-1 MOE 876 147073 49524 49535CACTGATCCTGC 1-10-1 MOE 842 147073 49672 49683 CACTGATCCTGC 1-10-1 MOE842 147074 49673 49684 CCACTGATCCTG 1-10-1 MOE 845 147036 50421 50432CCCAGTTCCCAG 1-10-1 MOE 849 147036 52292 52303 CCCAGTTCCCAG 1-10-1 MOE849 147037 52293 52304 GCCCAGTTCCCA 1-10-1 MOE 863 147036 52438 52449CCCAGTTCCCAG 1-10-1 MOE 849 147037 52439 52450 GCCCAGTTCCCA 1-10-1 MOE863 147034 53148 53159 CAGTTCCCAGCC 1-10-1 MOE 844 147034 53294 53305CAGTTCCCAGCC 1-10-1 MOE 844 147042 53445 53456 GGTCAAAAGGGC 1-10-1 MOE866 147043 53446 53457 TGGTCAAAAGGG 1-10-1 MOE 881 147044 53447 53458GTGGTCAAAAGG 1-10-1 MOE 869 147042 53591 53602 GGTCAAAAGGGC 1-10-1 MOE866 147030 53592 53603 TCCCAGCCTTGT 1-10-1 MOE 874 147043 53592 53603TGGTCAAAAGGG 1-10-1 MOE 881 147031 53593 53604 TTCCCAGCCTTG 1-10-1 MOE885 147044 53593 53604 GTGGTCAAAAGG 1-10-1 MOE 869 147030 53738 53749TCCCAGCCTTGT 1-10-1 MOE 874 147031 53739 53750 TTCCCAGCCTTG 1-10-1 MOE885 147040 53783 53794 GCCGCCCAGTTC 1-10-1 MOE 864 147041 53784 53795AGCCGCCCAGTT 1-10-1 MOE 834 147041 53930 53941 AGCCGCCCAGTT 1-10-1 MOE834 147042 55008 55019 GGTCAAAAGGGC 1-10-1 MOE 866 147043 55009 55020TGGTCAAAAGGG 1-10-1 MOE 881 147042 55154 55165 GGTCAAAAGGGC 1-10-1 MOE866 147043 55155 55166 TGGTCAAAAGGG 1-10-1 MOE 881 147058 55281 55292AATTTAATCCGA 1-10-1 MOE 830 147058 55427 55438 AATTTAATCCGA 1-10-1 MOE830 147019 55682 55693 TCGATCTCCTCG 1-10-1 MOE 877 147021 55684 55695TGTCGATCTCCT 1-10-1 MOE 882 147021 55830 55841 TGTCGATCTCCT 1-10-1 MOE882 147054 56275 56286 TAATCCGACTGT 1-10-1 MOE 871 147055 56276 56287TTAATCCGACTG 1-10-1 MOE 884 147056 56277 56288 TTTAATCCGACT 1-10-1 MOE887 147058 56279 56290 AATTTAATCCGA 1-10-1 MOE 830 147059 56280 56291CAATTTAATCCG 1-10-1 MOE 840 147060 56281 56292 GCAATTTAATCC 1-10-1 MOE861 147061 56282 56293 TGCAATTTAATC 1-10-1 MOE 879 147051 56418 56429TCCGACTGTGGT 1-10-1 MOE 875 147053 56420 56431 AATCCGACTGTG 1-10-1 MOE829 147054 56421 56432 TAATCCGACTGT 1-10-1 MOE 871 147055 56422 56433TTAATCCGACTG 1-10-1 MOE 884 147056 56423 56434 TTTAATCCGACT 1-10-1 MOE887 147057 56424 56435 ATTTAATCCGAC 1-10-1 MOE 839 147058 56425 56436AATTTAATCCGA 1-10-1 MOE 830 147061 56428 56439 TGCAATTTAATC 1-10-1 MOE879 147045 57118 57129 TGTGGTCAAAAG 1-10-1 MOE 883 147045 57264 57275TGTGGTCAAAAG 1-10-1 MOE 883 147046 57265 57276 CTGTGGTCAAAA 1-10-1 MOE858 147071 58028 58039 CTGATCCTGCAC 1-10-1 MOE 856 147071 58174 58185CTGATCCTGCAC 1-10-1 MOE 856 147043 61111 61122 TGGTCAAAAGGG 1-10-1 MOE881 147071 61130 61141 CTGATCCTGCAC 1-10-1 MOE 856 147020 61226 61237GTCGATCTCCTC 1-10-1 MOE 868 147043 61257 61268 TGGTCAAAAGGG 1-10-1 MOE881 147071 61276 61287 CTGATCCTGCAC 1-10-1 MOE 856 147035 61277 61288CCAGTTCCCAGC 1-10-1 MOE 847 147036 61278 61289 CCCAGTTCCCAG 1-10-1 MOE849 147037 61279 61290 GCCCAGTTCCCA 1-10-1 MOE 863 147038 61280 61291CGCCCAGTTCCC 1-10-1 MOE 855 147039 61281 61292 CCGCCCAGTTCC 1-10-1 MOE850 147040 61282 61293 GCCGCCCAGTTC 1-10-1 MOE 864 147071 61309 61320CTGATCCTGCAC 1-10-1 MOE 856 147020 61372 61383 GTCGATCTCCTC 1-10-1 MOE868 147034 61422 61433 CAGTTCCCAGCC 1-10-1 MOE 844 147035 61423 61434CCAGTTCCCAGC 1-10-1 MOE 847 147036 61424 61435 CCCAGTTCCCAG 1-10-1 MOE849 147037 61425 61436 GCCCAGTTCCCA 1-10-1 MOE 863 147038 61426 61437CGCCCAGTTCCC 1-10-1 MOE 855 147040 61428 61439 GCCGCCCAGTTC 1-10-1 MOE864 147071 61455 61466 CTGATCCTGCAC 1-10-1 MOE 856 147073 62003 62014CACTGATCCTGC 1-10-1 MOE 842 147073 62149 62160 CACTGATCCTGC 1-10-1 MOE842 147066 63065 63076 CCTGCACTGACG 1-10-1 MOE 851 147068 63067 63078ATCCTGCACTGA 1-10-1 MOE 838 147069 63146 63157 GATCCTGCACTG 1-10-1 MOE860 147062 63207 63218 CACTGACGAGTC 1-10-1 MOE 841 147066 63211 63222CCTGCACTGACG 1-10-1 MOE 851 147057 64054 64065 ATTTAATCCGAC 1-10-1 MOE839 147036 64538 64549 CCCAGTTCCCAG 1-10-1 MOE 849 147037 64539 64550GCCCAGTTCCCA 1-10-1 MOE 863 147037 64685 64696 GCCCAGTTCCCA 1-10-1 MOE863 147066 64864 64875 CCTGCACTGACG 1-10-1 MOE 851 147067 64865 64876TCCTGCACTGAC 1-10-1 MOE 876 147066 65010 65021 CCTGCACTGACG 1-10-1 MOE851 147067 65011 65022 TCCTGCACTGAC 1-10-1 MOE 876 147045 65017 65028TGTGGTCAAAAG 1-10-1 MOE 883 147045 65163 65174 TGTGGTCAAAAG 1-10-1 MOE883 147046 65164 65175 CTGTGGTCAAAA 1-10-1 MOE 858 147068 65408 65419ATCCTGCACTGA 1-10-1 MOE 838 147071 65411 65422 CTGATCCTGCAC 1-10-1 MOE856 147069 65549 65560 GATCCTGCACTG 1-10-1 MOE 860 147068 65554 65565ATCCTGCACTGA 1-10-1 MOE 838 147071 65557 65568 CTGATCCTGCAC 1-10-1 MOE856 147029 67741 67752 CCCAGCCTTGTC 1-10-1 MOE 848 147030 67742 67753TCCCAGCCTTGT 1-10-1 MOE 874 147031 67743 67754 TTCCCAGCCTTG 1-10-1 MOE885 147028 67886 67897 CCAGCCTTGTCG 1-10-1 MOE 846 147029 67887 67898CCCAGCCTTGTC 1-10-1 MOE 848 147030 67888 67899 TCCCAGCCTTGT 1-10-1 MOE874 147031 67889 67900 TTCCCAGCCTTG 1-10-1 MOE 885 147043 68867 68878TGGTCAAAAGGG 1-10-1 MOE 881 147044 68868 68879 GTGGTCAAAAGG 1-10-1 MOE869 147045 68869 68880 TGTGGTCAAAAG 1-10-1 MOE 883 147043 69013 69024TGGTCAAAAGGG 1-10-1 MOE 881 147044 69014 69025 GTGGTCAAAAGG 1-10-1 MOE869 147045 69015 69026 TGTGGTCAAAAG 1-10-1 MOE 883 147046 69016 69027CTGTGGTCAAAA 1-10-1 MOE 858 147071 69519 69530 CTGATCCTGCAC 1-10-1 MOE856 147072 69520 69531 ACTGATCCTGCA 1-10-1 MOE 832 147073 69521 69532CACTGATCCTGC 1-10-1 MOE 842 147071 69665 69676 CTGATCCTGCAC 1-10-1 MOE856 147072 69666 69677 ACTGATCCTGCA 1-10-1 MOE 832 147073 69667 69678CACTGATCCTGC 1-10-1 MOE 842 147074 69668 69679 CCACTGATCCTG 1-10-1 MOE845 147066 69869 69880 CCTGCACTGACG 1-10-1 MOE 851 147066 70015 70026CCTGCACTGACG 1-10-1 MOE 851 147023 70465 70476 CTTGTCGATCTC 1-10-1 MOE859 147023 70611 70622 CTTGTCGATCTC 1-10-1 MOE 859 147062 70615 70626CACTGACGAGTC 1-10-1 MOE 841 147063 70616 70627 GCACTGACGAGT 1-10-1 MOE862 147064 70617 70628 TGCACTGACGAG 1-10-1 MOE 880 147065 70618 70629CTGCACTGACGA 1-10-1 MOE 857 147066 70619 70630 CCTGCACTGACG 1-10-1 MOE851 147063 70762 70773 GCACTGACGAGT 1-10-1 MOE 862 147064 70763 70774TGCACTGACGAG 1-10-1 MOE 880 147065 70764 70775 CTGCACTGACGA 1-10-1 MOE857 147066 70765 70776 CCTGCACTGACG 1-10-1 MOE 851 147072 70998 71009ACTGATCCTGCA 1-10-1 MOE 832 147073 70999 71010 CACTGATCCTGC 1-10-1 MOE842 147072 71144 71155 ACTGATCCTGCA 1-10-1 MOE 832 147073 71145 71156CACTGATCCTGC 1-10-1 MOE 842 147074 71146 71157 CCACTGATCCTG 1-10-1 MOE845 147037 71351 71362 GCCCAGTTCCCA 1-10-1 MOE 863 147038 71352 71363CGCCCAGTTCCC 1-10-1 MOE 855 147039 71353 71364 CCGCCCAGTTCC 1-10-1 MOE850 147037 71497 71508 GCCCAGTTCCCA 1-10-1 MOE 863 147038 71498 71509CGCCCAGTTCCC 1-10-1 MOE 855 147039 71499 71510 CCGCCCAGTTCC 1-10-1 MOE850 147061 71641 71652 TGCAATTTAATC 1-10-1 MOE 879 147061 71787 71798TGCAATTTAATC 1-10-1 MOE 879

TABLE 17 Short antisense compounds targeted to SEQ ID NO: 11 and having1 or 2 mismatches 5′ 3′ SEQ ISIS Target Target Gapmer ID NO. Site SiteSequence (5′-3′) Motif NO 147022 177 188 TTGTCGATCTCC 1-10-1 MOE 886147023 178 189 CTTGTCGATCTC 1-10-1 MOE 859 147020 196 207 GTCGATCTCCTC1-10-1 MOE 868 147022 198 209 TTGTCGATCTCC 1-10-1 MOE 886 147024 200 211CCTTGTCGATCT 1-10-1 MOE 853 147026 202 213 AGCCTTGTCGAT 1-10-1 MOE 835147028 204 215 CCAGCCTTGTCG 1-10-1 MOE 846 147029 205 216 CCCAGCCTTGTC1-10-1 MOE 848 147030 206 217 TCCCAGCCTTGT 1-10-1 MOE 874 147036 212 223CCCAGTTCCCAG 1-10-1 MOE 849 147073 311 322 CACTGATCCTGC 1-10-1 MOE 842147046 327 338 CTGTGGTCAAAA 1-10-1 MOE 858 147047 328 339 ACTGTGGTCAAA1-10-1 MOE 833 147048 329 340 GACTGTGGTCAA 1-10-1 MOE 888 147049 330 341CGACTGTGGTCA 1-10-1 MOE 854 147050 331 342 CCGACTGTGGTC 1-10-1 MOE 889147051 332 343 TCCGACTGTGGT 1-10-1 MOE 875 147052 333 344 ATCCGACTGTGG1-10-1 MOE 837 147053 334 345 AATCCGACTGTG 1-10-1 MOE 829 147054 335 346TAATCCGACTGT 1-10-1 MOE 871 147055 336 347 TTAATCCGACTG 1-10-1 MOE 884147056 337 348 TTTAATCCGACT 1-10-1 MOE 887 147057 338 349 ATTTAATCCGAC1-10-1 MOE 839 147058 339 350 AATTTAATCCGA 1-10-1 MOE 830 147060 341 352GCAATTTAATCC 1-10-1 MOE 861 147061 342 353 TGCAATTTAATC 1-10-1 MOE 879147062 1024 1035 CACTGACGAGTC 1-10-1 MOE 841 147063 1025 1036GCACTGACGAGT 1-10-1 MOE 862 147068 1030 1041 ATCCTGCACTGA 1-10-1 MOE 838147071 1033 1044 CTGATCCTGCAC 1-10-1 MOE 856 147073 1035 1046CACTGATCCTGC 1-10-1 MOE 842 147074 1036 1047 CCACTGATCCTG 1-10-1 MOE 845147067 1091 1102 TCCTGCACTGAC 1-10-1 MOE 876 147024 1891 1902CCTTGTCGATCT 1-10-1 MOE 853 147026 1893 1904 AGCCTTGTCGAT 1-10-1 MOE 835147029 1896 1907 CCCAGCCTTGTC 1-10-1 MOE 848 147036 1903 1914CCCAGTTCCCAG 1-10-1 MOE 849 147039 1906 1917 CCGCCCAGTTCC 1-10-1 MOE 850147019 1994 2005 TCGATCTCCTCG 1-10-1 MOE 877 401385 2815 2828CCCAGTGGGTTTGA 2-10-2 MOE 890 147033 5265 5276 AGTTCCCAGCCT 1-10-1 MOE836 147033 5373 5384 AGTTCCCAGCCT 1-10-1 MOE 836 147060 7168 7179GCAATTTAATCC 1-10-1 MOE 861 147053 10527 10538 AATCCGACTGTG 1-10-1 MOE829 147053 10635 10646 AATCCGACTGTG 1-10-1 MOE 829 147070 11604 11615TGATCCTGCACT 1-10-1 MOE 878 147071 11612 11623 CTGATCCTGCAC 1-10-1 MOE856 147072 12294 12305 ACTGATCCTGCA 1-10-1 MOE 832 147072 12299 12310ACTGATCCTGCA 1-10-1 MOE 832 147052 12938 12949 ATCCGACTGTGG 1-10-1 MOE837 147052 13119 13130 ATCCGACTGTGG 1-10-1 MOE 837 147047 13158 13169ACTGTGGTCAAA 1-10-1 MOE 833 147048 13159 13170 GACTGTGGTCAA 1-10-1 MOE888 147049 13160 13171 CGACTGTGGTCA 1-10-1 MOE 854 147048 13340 13351GACTGTGGTCAA 1-10-1 MOE 888 147049 13341 13352 CGACTGTGGTCA 1-10-1 MOE854 147051 13343 13354 TCCGACTGTGGT 1-10-1 MOE 875 147061 13497 13508TGCAATTTAATC 1-10-1 MOE 879 147069 15562 15573 GATCCTGCACTG 1-10-1 MOE860 147068 15743 15754 ATCCTGCACTGA 1-10-1 MOE 838 147049 17181 17192CGACTGTGGTCA 1-10-1 MOE 854 147049 17349 17360 CGACTGTGGTCA 1-10-1 MOE854 147047 22438 22449 ACTGTGGTCAAA 1-10-1 MOE 833 147047 24322 24333ACTGTGGTCAAA 1-10-1 MOE 833 147045 24488 24499 TGTGGTCAAAAG 1-10-1 MOE883 147039 25064 25075 CCGCCCAGTTCC 1-10-1 MOE 850 147057 25508 25519ATTTAATCCGAC 1-10-1 MOE 839 147057 25676 25687 ATTTAATCCGAC 1-10-1 MOE839 147061 25680 25691 TGCAATTTAATC 1-10-1 MOE 879 147069 28731 28742GATCCTGCACTG 1-10-1 MOE 860 147052 30132 30143 ATCCGACTGTGG 1-10-1 MOE837 147052 30277 30288 ATCCGACTGTGG 1-10-1 MOE 837 147036 32085 32096CCCAGTTCCCAG 1-10-1 MOE 849 147072 32520 32531 ACTGATCCTGCA 1-10-1 MOE832 147071 33058 33069 CTGATCCTGCAC 1-10-1 MOE 856 147050 33125 33136CCGACTGTGGTC 1-10-1 MOE 889 147069 33204 33215 GATCCTGCACTG 1-10-1 MOE860 147050 33273 33284 CCGACTGTGGTC 1-10-1 MOE 889 147047 33319 33330ACTGTGGTCAAA 1-10-1 MOE 833 147050 33322 33333 CCGACTGTGGTC 1-10-1 MOE889 147052 33324 33335 ATCCGACTGTGG 1-10-1 MOE 837 147049 33469 33480CGACTGTGGTCA 1-10-1 MOE 854 147050 33470 33481 CCGACTGTGGTC 1-10-1 MOE889 147052 33472 33483 ATCCGACTGTGG 1-10-1 MOE 837 147047 33641 33652ACTGTGGTCAAA 1-10-1 MOE 833 147047 33789 33800 ACTGTGGTCAAA 1-10-1 MOE833 147059 35585 35596 CAATTTAATCCG 1-10-1 MOE 840 147021 36241 36252TGTCGATCTCCT 1-10-1 MOE 882 147073 37633 37644 CACTGATCCTGC 1-10-1 MOE842 147033 42529 42540 AGTTCCCAGCCT 1-10-1 MOE 836 147050 45401 45412CCGACTGTGGTC 1-10-1 MOE 889 147050 45549 45560 CCGACTGTGGTC 1-10-1 MOE889 147074 46125 46136 CCACTGATCCTG 1-10-1 MOE 845 147057 46313 46324ATTTAATCCGAC 1-10-1 MOE 839 147058 46462 46473 AATTTAATCCGA 1-10-1 MOE830 147058 47413 47424 AATTTAATCCGA 1-10-1 MOE 830 147058 47561 47572AATTTAATCCGA 1-10-1 MOE 830 147073 48221 48232 CACTGATCCTGC 1-10-1 MOE842 147073 48369 48380 CACTGATCCTGC 1-10-1 MOE 842 147028 48567 48578CCAGCCTTGTCG 1-10-1 MOE 846 147068 49199 49210 ATCCTGCACTGA 1-10-1 MOE838 147036 50273 50284 CCCAGTTCCCAG 1-10-1 MOE 849 147040 53929 53940GCCGCCCAGTTC 1-10-1 MOE 864 147047 54769 54780 ACTGTGGTCAAA 1-10-1 MOE833 147048 54770 54781 GACTGTGGTCAA 1-10-1 MOE 888 147047 54915 54926ACTGTGGTCAAA 1-10-1 MOE 833 147048 54916 54927 GACTGTGGTCAA 1-10-1 MOE888 147019 55828 55839 TCGATCTCCTCG 1-10-1 MOE 877 147047 56268 56279ACTGTGGTCAAA 1-10-1 MOE 833 147048 56269 56280 GACTGTGGTCAA 1-10-1 MOE888 147049 56270 56281 CGACTGTGGTCA 1-10-1 MOE 854 147050 56271 56282CCGACTGTGGTC 1-10-1 MOE 889 147051 56272 56283 TCCGACTGTGGT 1-10-1 MOE875 147052 56273 56284 ATCCGACTGTGG 1-10-1 MOE 837 147053 56274 56285AATCCGACTGTG 1-10-1 MOE 829 147056 56277 56288 TTTAATCCGACT 1-10-1 MOE887 147057 56278 56289 ATTTAATCCGAC 1-10-1 MOE 839 147047 56414 56425ACTGTGGTCAAA 1-10-1 MOE 833 147048 56415 56426 GACTGTGGTCAA 1-10-1 MOE888 147049 56416 56427 CGACTGTGGTCA 1-10-1 MOE 854 147050 56417 56428CCGACTGTGGTC 1-10-1 MOE 889 147052 56419 56430 ATCCGACTGTGG 1-10-1 MOE837 147057 56424 56435 ATTTAATCCGAC 1-10-1 MOE 839 147058 56425 56436AATTTAATCCGA 1-10-1 MOE 830 147059 56426 56437 CAATTTAATCCG 1-10-1 MOE840 147060 56427 56438 GCAATTTAATCC 1-10-1 MOE 861 147046 57119 57130CTGTGGTCAAAA 1-10-1 MOE 858 147071 58174 58185 CTGATCCTGCAC 1-10-1 MOE856 147071 61130 61141 CTGATCCTGCAC 1-10-1 MOE 856 147034 61276 61287CAGTTCCCAGCC 1-10-1 MOE 844 147071 61309 61320 CTGATCCTGCAC 1-10-1 MOE856 147039 61427 61438 CCGCCCAGTTCC 1-10-1 MOE 850 147071 61455 61466CTGATCCTGCAC 1-10-1 MOE 856 147073 62003 62014 CACTGATCCTGC 1-10-1 MOE842 147062 63061 63072 CACTGACGAGTC 1-10-1 MOE 841 147068 63213 63224ATCCTGCACTGA 1-10-1 MOE 838 147069 63292 63303 GATCCTGCACTG 1-10-1 MOE860 147057 64054 64065 ATTTAATCCGAC 1-10-1 MOE 839 147057 64200 64211ATTTAATCCGAC 1-10-1 MOE 839 147070 64427 64438 TGATCCTGCACT 1-10-1 MOE878 147070 64573 64584 TGATCCTGCACT 1-10-1 MOE 878 147036 64684 64695CCCAGTTCCCAG 1-10-1 MOE 849 147046 65018 65029 CTGTGGTCAAAA 1-10-1 MOE858 147071 65557 65568 CTGATCCTGCAC 1-10-1 MOE 856 147069 65695 65706GATCCTGCACTG 1-10-1 MOE 860 147047 66163 66174 ACTGTGGTCAAA 1-10-1 MOE833 147047 66309 66320 ACTGTGGTCAAA 1-10-1 MOE 833 147028 67740 67751CCAGCCTTGTCG 1-10-1 MOE 846 147046 68870 68881 CTGTGGTCAAAA 1-10-1 MOE858 147047 68871 68882 ACTGTGGTCAAA 1-10-1 MOE 833 147048 68872 68883GACTGTGGTCAA 1-10-1 MOE 888 147049 68873 68884 CGACTGTGGTCA 1-10-1 MOE854 147047 69017 69028 ACTGTGGTCAAA 1-10-1 MOE 833 147048 69018 69029GACTGTGGTCAA 1-10-1 MOE 888 147049 69019 69030 CGACTGTGGTCA 1-10-1 MOE854 147071 69519 69530 CTGATCCTGCAC 1-10-1 MOE 856 147073 69521 69532CACTGATCCTGC 1-10-1 MOE 842 147071 69665 69676 CTGATCCTGCAC 1-10-1 MOE856 147072 69666 69677 ACTGATCCTGCA 1-10-1 MOE 832 147024 70466 70477CCTTGTCGATCT 1-10-1 MOE 853 147024 70612 70623 CCTTGTCGATCT 1-10-1 MOE853 147062 70761 70772 CACTGACGAGTC 1-10-1 MOE 841 147072 70998 71009ACTGATCCTGCA 1-10-1 MOE 832 147073 70999 71010 CACTGATCCTGC 1-10-1 MOE842 147072 71144 71155 ACTGATCCTGCA 1-10-1 MOE 832 147073 71145 71156CACTGATCCTGC 1-10-1 MOE 842 147048 71366 71377 GACTGTGGTCAA 1-10-1 MOE888 147048 71512 71523 GACTGTGGTCAA 1-10-1 MOE 888

TABLE 18 Short Antisense Compounds targeted to SEQ ID NO: 12 5′ 3′ SeqISIS Target Target Sequence Gapmer ID NO. Site Site (5′-3′) Motif NO398163 20 31 ATGTCAACCGGC 1-10-1 MOE 908 384545 23 34 CAAGTAGGATGT1-10-1 MOE 951 147705 159 170 CGGTTTTTGTTC 1-10-1 MOE 1002 147703 245256 TGGCTTCATGTC 1-10-1 MOE 971 398090 283 296 TTGTTCTTAGGAAG 2-10-2 MOE972 147704 285 296 TTGTTCTTAGGA 1-10-1 MOE 1012 147705 291 302CGGTTTTTGTTC 1-10-1 MOE 1002 147709 311 322 CCATTTTTATCA 1-10-1 MOE 978147733 349 360 TTCTTGATGTCC 1-10-1 MOE 891 147707 360 371 TAGTCATTATCT1-10-1 MOE 977 147708 366 377 TTGATATAGTCA 1-10-1 MOE 997 390030 381 392TTTATAAAACTG 1-10-1 MOE 1074 147709 386 397 CCATTTTTATCA 1-10-1 MOE 978147081 393 404 GCTCCTTCCACT 1-10-1 MOE 1006 398091 393 406GGGCTTCTTCCATT 2-10-2 MOE 979 398166 395 406 GGGCTTCTTCCA 1-10-1 MOE1070 147709 418 429 CCATTTTTATCA 1-10-1 MOE 978 147711 425 436AAGGGCCCTGGG 1-10-1 MOE 1040 147712 461 472 ACACCATCTCCC 1-10-1 MOE 1005147713 466 477 CTCCCACACCAT 1-10-1 MOE 985 147714 471 482 TTCTGCTCCCAC1-10-1 MOE 986 147715 496 507 GTTGAGCATGAC 1-10-1 MOE 1077 147716 521532 TTAACGAGCCTT 1-10-1 MOE 949 147717 574 585 ATCTTCAGAGAT 1-10-1 MOE996 147717 607 618 ATCTTCAGAGAT 1-10-1 MOE 996 147708 612 623TTGATATAGTCA 1-10-1 MOE 997 147718 621 632 TAATATGACTTG 1-10-1 MOE 998147746 625 636 TAAAAACAACAA 1-10-1 MOE 1073 398167 704 715 CAGGCCATGTGG1-10-1 MOE 1059 398092 705 718 AGTCAGGCCATGTG 2-10-2 MOE 1060 147723 715726 GACTCCAAAGTC 1-10-1 MOE 892 398093 758 771 TCGGACTTTGAAAA 2-10-2 MOE1009 398168 760 771 TCGGACTTTGAA 1-10-1 MOE 1008 147738 780 791TGGGTGGCCGGG 1-10-1 MOE 1069 398094 848 861 ATCAGCCAGACAGA 2-10-2 MOE1010 398169 849 860 TCAGCCAGACAG 1-10-1 MOE 909 398164 873 884TTGTCGATCTGC 1-10-1 MOE 1014 147735 973 984 GGAGAAGCGCAG 1-10-1 MOE 1016147737 984 995 ACAGCCAGGTAG 1-10-1 MOE 1067 368369 1025 1040TCCTGCACTGACG 3-10-3 MOE 893 AGT 368372 1031 1046 CACTGATCCTGCA 3-10-3MOE 894 CTG 368353 1033 1046 CACTGATCCTGCAC 2-10-2 MOE 1007 368354 10351048 TCCACTGATCCTGC 2-10-2 MOE 1024 368388 1035 1050 CTTCCACTGATCC3-10-3 MOE 895 TTA 368355 1036 1049 TTCCACTGATCCTG 2-10-2 MOE 1025368356 1037 1050 CTTCCACTGATCCT 2-10-2 MOE 1027 368376 1037 1052TCCTTCCACTGAT 3-10-3 MOE 1028 CCT 147076 1038 1049 TTCCACTGATCC 1-10-1MOE 1029 368357 1038 1051 CCTTCCACTGATCC 2-10-2 MOE 1046 147077 10391050 CTTCCACTGATC 1-10-1 MOE 1047 368358 1039 1052 TCCTTCCACTGATC 2-10-2MOE 1031 368378 1039 1054 GCTCCTTCCACTG 3-10-3 MOE 1032 ATC 368359 10411054 GCTCCTTCCACTGA 2-10-2 MOE 1033 147080 1042 1053 CTCCTTCCACTG 1-10-1MOE 1021 147081 1043 1054 GCTCCTTCCACT 1-10-1 MOE 1006 368360 1043 1056AAGCTCCTTCCACT 2-10-2 MOE 1035 368380 1043 1058 GAAAGCTCCTTCC 3-10-3 MOE896 ACT 147082 1044 1055 AGCTCCTTCCAC 1-10-1 MOE 1036 368381 1045 1060GGGAAAGCTCCTT 3-10-3 MOE 1037 CCA 147739 1107 1118 CGTTTGGGTGGC 1-10-1MOE 1023 147741 1165 1176 CACCCACTGGTG 1-10-1 MOE 1055 398097 1194 1207GGCAGTCTTTATCC 2-10-2 MOE 897 147742 1273 1284 AACTTCAGTGTC 1-10-1 MOE1041 147743 1388 1399 AGGGCTTCCAGT 1-10-1 MOE 1042 147744 1392 1403AGGAAGGGCTTC 1-10-1 MOE 1043 147745 1398 1409 TTGACCAGGAAG 1-10-1 MOE1058 398157 1455 1468 GGAAACATACCCTG 2-10-2 MOE 1045 398167 1475 1486CAGGCCATGTGG 1-10-1 MOE 1059 398092 1476 1489 AGTCAGGCCATGTG 2-10-2 MOE1060 368357 1596 1609 CCTTCCACTGATCC 2-10-2 MOE 1046 398160 1691 1704GAATAGGTTAAGGC 2-10-2 MOE 1048 398163 1711 1722 ATGTCAACCGGC 1-10-1 MOE908 147746 1750 1761 TAAAAACAACAA 1-10-1 MOE 1073 389949 1777 1788GCGCGAGCCCGA 1-10-1 MOE 1061 398161 1790 1803 AACAATGTGTTGTA 2-10-2 MOE1049 147746 1799 1810 TAAAAACAACAA 1-10-1 MOE 1073 398163 1819 1830ATGTCAACCGGC 1-10-1 MOE 908 389950 1848 1859 CCCTGAAGGTTC 1-10-1 MOE1063 398164 1889 1900 TTGTCGATCTGC 1-10-1 MOE 1014 147702 1917 1928CTGGTAAATAGC 1-10-1 MOE 898 147088 1971 1982 CCCTCTACACCA 1-10-1 MOE1050 398102 2003 2016 CTACCTGAGGATTT 2-10-2 MOE 899 398103 2010 2023CCCAGTACTACCTG 2-10-2 MOE 900 147737 2386 2397 ACAGCCAGGTAG 1-10-1 MOE1067 398095 2407 2420 CATCAGCAAGAGGC 2-10-2 MOE 1011 398106 2441 2454TGGAAAACTGCACC 2-10-2 MOE 1068 147745 2497 2508 TTGACCAGGAAG 1-10-1 MOE1058 147712 2499 2510 ACACCATCTCCC 1-10-1 MOE 1005 147712 2607 2618ACACCATCTCCC 1-10-1 MOE 1005 147745 2689 2700 TTGACCAGGAAG 1-10-1 MOE1058 398167 2706 2717 CAGGCCATGTGG 1-10-1 MOE 1059 398092 2707 2720AGTCAGGCCATGTG 2-10-2 MOE 1060 398166 2966 2977 GGGCTTCTTCCA 1-10-1 MOE1070 147091 2992 3003 GTTCCCTCTACA 1-10-1 MOE 1004 147092 2993 3004TGTTCCCTCTAC 1-10-1 MOE 901 389949 3008 3019 GCGCGAGCCCGA 1-10-1 MOE1061 147087 3149 3160 CCTCTACACCAG 1-10-1 MOE 982 147088 3150 3161CCCTCTACACCA 1-10-1 MOE 1050 398113 3160 3173 AGGAGGTTAAACCA 2-10-2 MOE905 147087 3257 3268 CCTCTACACCAG 1-10-1 MOE 982 147088 3258 3269CCCTCTACACCA 1-10-1 MOE 1050 147737 3591 3602 ACAGCCAGGTAG 1-10-1 MOE1067 147737 3617 3628 ACAGCCAGGTAG 1-10-1 MOE 1067 147079 3637 3648TCCTTCCACTGA 1-10-1 MOE 1001 147080 3638 3649 CTCCTTCCACTG 1-10-1 MOE1021 398095 3638 3651 CATCAGCAAGAGGC 2-10-2 MOE 1011 398106 3672 3685TGGAAAACTGCACC 2-10-2 MOE 1068 398107 3678 3691 TATTCCTGGAAAAC 2-10-2MOE 902 147691 3806 3817 GAGGTGGGAAAA 1-10-1 MOE 966 147683 3848 3859GCTTACGATTGT 1-10-1 MOE 922 147738 3853 3864 TGGGTGGCCGGG 1-10-1 MOE1069 398167 3926 3937 CAGGCCATGTGG 1-10-1 MOE 1059 398109 3945 3958CAAGAAGTGTGGTT 2-10-2 MOE 903 398167 4034 4045 CAGGCCATGTGG 1-10-1 MOE1059 398110 4083 4096 GTTCCCTTTGCAGG 2-10-2 MOE 952 398111 4168 4181GTGAAAATGCTGGC 2-10-2 MOE 904 147706 4238 4249 GCTGACATCTCG 1-10-1 MOE1071 398112 4282 4295 CAGCCTGGCACCTA 2-10-2 MOE 1072 147746 4315 4326TAAAAACAACAA 1-10-1 MOE 1073 398113 4391 4404 AGGAGGTTAAACCA 2-10-2 MOE905 398115 4484 4497 AGTAAATATTGGCT 2-10-2 MOE 1076 390030 4491 4502TTTATAAAACTG 1-10-1 MOE 1074 390030 4537 4548 TTTATAAAACTG 1-10-1 MOE1074 147703 5034 5045 TGGCTTCATGTC 1-10-1 MOE 971 147684 5035 5046ACCCAGTCAGGG 1-10-1 MOE 964 398125 5075 5088 CAGTAAGGAATTTT 2-10-2 MOE913 147696 5083 5094 TGGATGATTGGC 1-10-1 MOE 906 147684 5143 5154ACCCAGTCAGGG 1-10-1 MOE 964 147712 5366 5377 ACACCATCTCCC 1-10-1 MOE1005 147714 5416 5427 TTCTGCTCCCAC 1-10-1 MOE 986 398128 5443 5456CTAAATTTAGTTCA 2-10-2 MOE 911 147712 5474 5485 ACACCATCTCCC 1-10-1 MOE1005 147746 5498 5509 TAAAAACAACAA 1-10-1 MOE 1073 147714 5524 5535TTCTGCTCCCAC 1-10-1 MOE 986 147736 5600 5611 AGGTAGGAGAAG 1-10-1 MOE 963147085 5762 5773 TCTACACCAGGT 1-10-1 MOE 961 147679 5825 5836CAAAAGGATCCC 1-10-1 MOE 907 390030 6803 6814 TTTATAAAACTG 1-10-1 MOE1074 398142 6885 6898 CCAGCACACTGGAA 2-10-2 MOE 923 398142 6994 7007CCAGCACACTGGAA 2-10-2 MOE 923 398166 7306 7317 GGGCTTCTTCCA 1-10-1 MOE1070 147684 7551 7562 ACCCAGTCAGGG 1-10-1 MOE 964 147085 8308 8319TCTACACCAGGT 1-10-1 MOE 961 147085 8416 8427 TCTACACCAGGT 1-10-1 MOE 961398163 8473 8484 ATGTCAACCGGC 1-10-1 MOE 908 147718 8523 8534TAATATGACTTG 1-10-1 MOE 998 147718 8631 8642 TAATATGACTTG 1-10-1 MOE 998147691 8806 8817 GAGGTGGGAAAA 1-10-1 MOE 966 147728 8835 8846GCCAGACAGAAG 1-10-1 MOE 1013 147728 8943 8954 GCCAGACAGAAG 1-10-1 MOE1013 398169 8946 8957 TCAGCCAGACAG 1-10-1 MOE 909 147742 9060 9071AACTTCAGTGTC 1-10-1 MOE 1041 404136 9162 9175 TAAGTGTCCCTTTG 2-10-2 MOE910 147746 9963 9974 TAAAAACAACAA 1-10-1 MOE 1073 147746 9966 9977TAAAAACAACAA 1-10-1 MOE 1073 147746 9969 9980 TAAAAACAACAA 1-10-1 MOE1073 147746 9991 10002 TAAAAACAACAA 1-10-1 MOE 1073 147746 10071 10082TAAAAACAACAA 1-10-1 MOE 1073 147746 10074 10085 TAAAAACAACAA 1-10-1 MOE1073 147746 10077 10088 TAAAAACAACAA 1-10-1 MOE 1073 390030 10170 10181TTTATAAAACTG 1-10-1 MOE 1074 147084 10220 10231 CTACACCAGGTC 1-10-1 MOE993 390030 10278 10289 TTTATAAAACTG 1-10-1 MOE 1074 147085 10329 10340TCTACACCAGGT 1-10-1 MOE 961 147711 10684 10695 AAGGGCCCTGGG 1-10-1 MOE1040 147711 10792 10803 AAGGGCCCTGGG 1-10-1 MOE 1040 398128 11333 11346CTAAATTTAGTTCA 2-10-2 MOE 911 147707 11960 11971 TAGTCATTATCT 1-10-1 MOE977 147707 11965 11976 TAGTCATTATCT 1-10-1 MOE 977 147090 12013 12024TTCCCTCTACAC 1-10-1 MOE 955 398096 12146 12159 GGAGAAGCGCAGCT 2-10-2 MOE1015 398166 12214 12225 GGGCTTCTTCCA 1-10-1 MOE 1070 398135 12308 12321GACTACATTTTACA 2-10-2 MOE 912 147741 12389 12400 CACCCACTGGTG 1-10-1 MOE1055 398125 12431 12444 CAGTAAGGAATTTT 2-10-2 MOE 913 147714 12585 12596TTCTGCTCCCAC 1-10-1 MOE 986 147718 12594 12605 TAATATGACTTG 1-10-1 MOE998 398125 12612 12625 CAGTAAGGAATTTT 2-10-2 MOE 913 147737 12803 12814ACAGCCAGGTAG 1-10-1 MOE 1067 147746 12876 12887 TAAAAACAACAA 1-10-1 MOE1073 147691 12900 12911 GAGGTGGGAAAA 1-10-1 MOE 966 398137 13111 13124TGTGTCCCTCAGTC 2-10-2 MOE 914 398138 13254 13267 AACATCAAGCTTGA 2-10-2MOE 931 398137 13292 13305 TGTGTCCCTCAGTC 2-10-2 MOE 914 398138 1343513448 AACATCAAGCTTGA 2-10-2 MOE 931 389764 14020 14031 CTGCAACATGAT1-9-2 MOE 1018 389948 14067 14078 CCGTTGGACCCC 1-10-1 MOE 915 38994814248 14259 CCGTTGGACCCC 1-10-1 MOE 915 147738 14279 14290 TGGGTGGCCGGG1-10-1 MOE 1069 147698 14572 14583 CCCGCCACCACC 1-10-1 MOE 928 14771714750 14761 ATCTTCAGAGAT 1-10-1 MOE 996 147717 14932 14943 ATCTTCAGAGAT1-10-1 MOE 996 398167 15374 15385 CAGGCCATGTGG 1-10-1 MOE 1059 14773616444 16455 AGGTAGGAGAAG 1-10-1 MOE 963 147746 16510 16521 TAAAAACAACAA1-10-1 MOE 1073 147738 16590 16601 TGGGTGGCCGGG 1-10-1 MOE 1069 14774616676 16687 TAAAAACAACAA 1-10-1 MOE 1073 398167 16797 16808 CAGGCCATGTGG1-10-1 MOE 1059 398144 16911 16924 GACAGCTTCTATAA 2-10-2 MOE 916 38976417096 17107 CTGCAACATGAT 1-9-2 MOE 1018 147709 17238 17249 CCATTTTTATCA1-10-1 MOE 978 147709 17406 17417 CCATTTTTATCA 1-10-1 MOE 978 14769517466 17477 TCATTCCCCACT 1-10-1 MOE 984 147746 17497 17508 TAAAAACAACAA1-10-1 MOE 1073 147088 17539 17550 CCCTCTACACCA 1-10-1 MOE 1050 14771117808 17819 AAGGGCCCTGGG 1-10-1 MOE 1040 147711 17976 17987 AAGGGCCCTGGG1-10-1 MOE 1040 398139 18049 18062 AGTGACTGACCACA 2-10-2 MOE 917 39813918217 18230 AGTGACTGACCACA 2-10-2 MOE 917 398140 18596 18609GTAGCATAGAGCCT 2-10-2 MOE 918 398140 18764 18777 GTAGCATAGAGCCT 2-10-2MOE 918 398167 18927 18938 CAGGCCATGTGG 1-10-1 MOE 1059 398141 1894718960 CAGATCTTGTCAAG 2-10-2 MOE 919 398167 19095 19106 CAGGCCATGTGG1-10-1 MOE 1059 398141 19115 19128 CAGATCTTGTCAAG 2-10-2 MOE 919 14774619207 19218 TAAAAACAACAA 1-10-1 MOE 1073 147711 19508 19519 AAGGGCCCTGGG1-10-1 MOE 1040 147729 19554 19565 GTAAGAGGCAGG 1-10-1 MOE 920 14771819617 19628 TAATATGACTTG 1-10-1 MOE 998 390030 19618 19629 TTTATAAAACTG1-10-1 MOE 1074 147701 19671 19682 CCATGGCGGGAC 1-10-1 MOE 921 14771119676 19687 AAGGGCCCTGGG 1-10-1 MOE 1040 147718 19785 19796 TAATATGACTTG1-10-1 MOE 998 147079 20515 20526 TCCTTCCACTGA 1-10-1 MOE 1001 38976420620 20631 CTGCAACATGAT 1-9-2 MOE 1018 398142 20653 20666CCAGCACACTGGAA 2-10-2 MOE 923 147078 20682 20693 CCTTCCACTGAT 1-10-1 MOE1044 147079 20683 20694 TCCTTCCACTGA 1-10-1 MOE 1001 147080 20704 20715CTCCTTCCACTG 1-10-1 MOE 1021 147081 20705 20716 GCTCCTTCCACT 1-10-1 MOE1006 389965 20788 20799 CTGCAACATGAT 1-10-1 MOE 1018 147746 20870 20881TAAAAACAACAA 1-10-1 MOE 1073 147746 21038 21049 TAAAAACAACAA 1-10-1 MOE1073 147717 21080 21091 ATCTTCAGAGAT 1-10-1 MOE 996 147076 21222 21233TTCCACTGATCC 1-10-1 MOE 1029 398094 21441 21454 ATCAGCCAGACAGA 2-10-2MOE 1010 147746 21633 21644 TAAAAACAACAA 1-10-1 MOE 1073 147738 2188421895 TGGGTGGCCGGG 1-10-1 MOE 1069 147683 21939 21950 GCTTACGATTGT1-10-1 MOE 922 147743 22213 22224 AGGGCTTCCAGT 1-10-1 MOE 1042 14773622759 22770 AGGTAGGAGAAG 1-10-1 MOE 963 147736 22927 22938 AGGTAGGAGAAG1-10-1 MOE 963 398142 23008 23021 CCAGCACACTGGAA 2-10-2 MOE 923 39814723784 23797 CTACAGGACAATAC 2-10-2 MOE 957 398147 23952 23965CTACAGGACAATAC 2-10-2 MOE 957 147713 24434 24445 CTCCCACACCAT 1-10-1 MOE985 389965 24543 24554 CTGCAACATGAT 1-10-1 MOE 1018 147713 24602 24613CTCCCACACCAT 1-10-1 MOE 985 389965 24711 24722 CTGCAACATGAT 1-10-1 MOE1018 147746 25384 25395 TAAAAACAACAA 1-10-1 MOE 1073 398143 25505 25518GTCAGTCCCAGCTA 2-10-2 MOE 924 147691 25610 25621 GAGGTGGGAAAA 1-10-1 MOE966 398130 25672 25685 TTAGTATGACAGCT 2-10-2 MOE 925 147746 25810 25821TAAAAACAACAA 1-10-1 MOE 1073 147746 25978 25989 TAAAAACAACAA 1-10-1 MOE1073 147746 26172 26183 TAAAAACAACAA 1-10-1 MOE 1073 398151 26718 26731TCAGTGTAGGAAGA 2-10-2 MOE 926 147728 26917 26928 GCCAGACAGAAG 1-10-1 MOE1013 398152 27708 27721 TGAATATACAGATG 2-10-2 MOE 927 147698 28629 28640CCCGCCACCACC 1-10-1 MOE 928 389965 28714 28725 CTGCAACATGAT 1-10-1 MOE1018 389764 28714 28725 CTGCAACATGAT 1-9-2 MOE 1018 389764 28861 28872CTGCAACATGAT 1-9-2 MOE 1018 390030 29945 29956 TTTATAAAACTG 1-10-1 MOE1074 147744 30654 30665 AGGAAGGGCTTC 1-10-1 MOE 1043 147093 30836 30847TTGTTCCCTCTA 1-10-1 MOE 929 147746 30957 30968 TAAAAACAACAA 1-10-1 MOE1073 147746 31105 31116 TAAAAACAACAA 1-10-1 MOE 1073 390030 31477 31488TTTATAAAACTG 1-10-1 MOE 1074 384545 31829 31840 CAAGTAGGATGT 1-10-1 MOE951 384545 31977 31988 CAAGTAGGATGT 1-10-1 MOE 951 401382 32094 32107TCTACCTGAGTCCA 2-10-2 MOE 930 147089 32387 32398 TCCCTCTACACC 1-10-1 MOE956 389950 32949 32960 CCCTGAAGGTTC 1-10-1 MOE 1063 398165 33002 33013GTTCTTAGGAAG 1-10-1 MOE 968 147081 33073 33084 GCTCCTTCCACT 1-10-1 MOE1006 147082 33074 33085 AGCTCCTTCCAC 1-10-1 MOE 1036 389950 33097 33108CCCTGAAGGTTC 1-10-1 MOE 1063 147736 33160 33171 AGGTAGGAGAAG 1-10-1 MOE963 147081 33221 33232 GCTCCTTCCACT 1-10-1 MOE 1006 368360 33221 33234AAGCTCCTTCCACT 2-10-2 MOE 1035 147082 33222 33233 AGCTCCTTCCAC 1-10-1MOE 1036 398138 33244 33257 AACATCAAGCTTGA 2-10-2 MOE 931 147746 3325033261 TAAAAACAACAA 1-10-1 MOE 1073 398138 33392 33405 AACATCAAGCTTGA2-10-2 MOE 931 401383 33588 33601 GATCACCTTCAGAG 2-10-2 MOE 932 14774633886 33897 TAAAAACAACAA 1-10-1 MOE 1073 147746 34606 34617 TAAAAACAACAA1-10-1 MOE 1073 398165 34704 34715 GTTCTTAGGAAG 1-10-1 MOE 968 14771734745 34756 ATCTTCAGAGAT 1-10-1 MOE 996 147746 34754 34765 TAAAAACAACAA1-10-1 MOE 1073 398165 34852 34863 GTTCTTAGGAAG 1-10-1 MOE 968 14771734893 34904 ATCTTCAGAGAT 1-10-1 MOE 996 401384 34905 34918TGAACACATCACTA 2-10-2 MOE 933 147738 35391 35402 TGGGTGGCCGGG 1-10-1 MOE1069 147736 35396 35407 AGGTAGGAGAAG 1-10-1 MOE 963 147738 35539 35550TGGGTGGCCGGG 1-10-1 MOE 1069 147691 35554 35565 GAGGTGGGAAAA 1-10-1 MOE966 147691 35702 35713 GAGGTGGGAAAA 1-10-1 MOE 966 147746 35814 35825TAAAAACAACAA 1-10-1 MOE 1073 401385 36109 36122 CCCAGTGGGTTTGA 2-10-2MOE 890 147691 36360 36371 GAGGTGGGAAAA 1-10-1 MOE 966 147746 3641636427 TAAAAACAACAA 1-10-1 MOE 1073 147731 36620 36631 TTTCCTCTTGTC1-10-1 MOE 934 147714 37881 37892 TTCTGCTCCCAC 1-10-1 MOE 986 14771438029 38040 TTCTGCTCCCAC 1-10-1 MOE 986 147681 38512 38523 ATGTCATTAAAC1-10-1 MOE 965 401386 38516 38529 TAATTGATGTCAAT 2-10-2 MOE 935 40138738518 38531 AGTAATTGATGTCA 2-10-2 MOE 936 401388 38520 38533ACAGTAATTGATGT 2-10-2 MOE 937 401389 38522 38535 TTACAGTAATTGAT 2-10-2MOE 938 401390 38524 38537 ACTTACAGTAATTG 2-10-2 MOE 939 401391 3852638539 AGACTTACAGTAAT 2-10-2 MOE 940 401392 38528 38541 TCAGACTTACAGTA2-10-2 MOE 941 401393 38530 38543 AATCAGACTTACAG 2-10-2 MOE 942 40139438532 38545 TGAATCAGACTTAC 2-10-2 MOE 943 401395 38534 38547AATGAATCAGACTT 2-10-2 MOE 944 147738 38909 38920 TGGGTGGCCGGG 1-10-1 MOE1069 147738 39057 39068 TGGGTGGCCGGG 1-10-1 MOE 1069 390030 39249 39260TTTATAAAACTG 1-10-1 MOE 1074 390030 39397 39408 TTTATAAAACTG 1-10-1 MOE1074 401396 39488 39501 TGCAGGATGTTGAG 2-10-2 MOE 945 147717 39545 39556ATCTTCAGAGAT 1-10-1 MOE 996 147746 39641 39652 TAAAAACAACAA 1-10-1 MOE1073 147717 39693 39704 ATCTTCAGAGAT 1-10-1 MOE 996 147746 39729 39740TAAAAACAACAA 1-10-1 MOE 1073 147746 39877 39888 TAAAAACAACAA 1-10-1 MOE1073 147746 40185 40196 TAAAAACAACAA 1-10-1 MOE 1073 147746 40478 40489TAAAAACAACAA 1-10-1 MOE 1073 398166 40589 40600 GGGCTTCTTCCA 1-10-1 MOE1070 147735 40662 40673 GGAGAAGCGCAG 1-10-1 MOE 1016 147746 40706 40717TAAAAACAACAA 1-10-1 MOE 1073 398166 40737 40748 GGGCTTCTTCCA 1-10-1 MOE1070 147746 40854 40865 TAAAAACAACAA 1-10-1 MOE 1073 401397 41012 41025CTGGTCAGCATTGA 2-10-2 MOE 946 147718 41070 41081 TAATATGACTTG 1-10-1 MOE998 147718 41218 41229 TAATATGACTTG 1-10-1 MOE 998 147717 41221 41232ATCTTCAGAGAT 1-10-1 MOE 996 147717 41369 41380 ATCTTCAGAGAT 1-10-1 MOE996 147717 41599 41610 ATCTTCAGAGAT 1-10-1 MOE 996 147717 41747 41758ATCTTCAGAGAT 1-10-1 MOE 996 401398 41768 41781 CAAAGTCCCTTAGC 2-10-2 MOE947 390030 42056 42067 TTTATAAAACTG 1-10-1 MOE 1074 398153 42157 42170ATTTCTCTTACAGG 2-10-2 MOE 948 398153 42305 42318 ATTTCTCTTACAGG 2-10-2MOE 948 147710 42691 42702 TATAGCTCCTCT 1-10-1 MOE 994 147079 4332243333 TCCTTCCACTGA 1-10-1 MOE 1001 147080 43323 43334 CTCCTTCCACTG1-10-1 MOE 1021 147716 43477 43488 TTAACGAGCCTT 1-10-1 MOE 949 14774643992 44003 TAAAAACAACAA 1-10-1 MOE 1073 147736 44137 44148 AGGTAGGAGAAG1-10-1 MOE 963 384545 44242 44253 CAAGTAGGATGT 1-10-1 MOE 951 14768744354 44365 CGACACGGGAAC 1-10-1 MOE 950 384545 44390 44401 CAAGTAGGATGT1-10-1 MOE 951 398110 44713 44726 GTTCCCTTTGCAGG 2-10-2 MOE 952 14770545092 45103 CGGTTTTTGTTC 1-10-1 MOE 1002 147705 45240 45251 CGGTTTTTGTTC1-10-1 MOE 1002 147074 45977 45988 CCACTGATCCTG 1-10-1 MOE 845 14707545978 45989 TCCACTGATCCT 1-10-1 MOE 1026 147076 45979 45990 TTCCACTGATCC1-10-1 MOE 1029 147076 46127 46138 TTCCACTGATCC 1-10-1 MOE 1029 40139946247 46260 ATTAGCCATATCTC 2-10-2 MOE 953 147705 46555 46566CGGTTTTTGTTC 1-10-1 MOE 1002 147714 46685 46696 TTCTGCTCCCAC 1-10-1 MOE986 147705 46703 46714 CGGTTTTTGTTC 1-10-1 MOE 1002 390030 46859 46870TTTATAAAACTG 1-10-1 MOE 1074 390030 46933 46944 TTTATAAAACTG 1-10-1 MOE1074 147681 46984 46995 ATGTCATTAAAC 1-10-1 MOE 965 390030 47007 47018TTTATAAAACTG 1-10-1 MOE 1074 147746 47023 47034 TAAAAACAACAA 1-10-1 MOE1073 390030 47081 47092 TTTATAAAACTG 1-10-1 MOE 1074 147681 47132 47143ATGTCATTAAAC 1-10-1 MOE 965 147746 47171 47182 TAAAAACAACAA 1-10-1 MOE1073 401400 47411 47424 AGCATTCAGCAGTG 2-10-2 MOE 954 147746 47461 47472TAAAAACAACAA 1-10-1 MOE 1073 147086 47608 47619 CTCTACACCAGG 1-10-1 MOE969 147087 47609 47620 CCTCTACACCAG 1-10-1 MOE 982 147088 47610 47621CCCTCTACACCA 1-10-1 MOE 1050 147090 47612 47623 TTCCCTCTACAC 1-10-1 MOE955 147691 47729 47740 GAGGTGGGAAAA 1-10-1 MOE 966 147086 47756 47767CTCTACACCAGG 1-10-1 MOE 969 147088 47758 47769 CCCTCTACACCA 1-10-1 MOE1050 147089 47759 47770 TCCCTCTACACC 1-10-1 MOE 956 390030 47847 47858TTTATAAAACTG 1-10-1 MOE 1074 390030 47995 48006 TTTATAAAACTG 1-10-1 MOE1074 147691 48393 48404 GAGGTGGGAAAA 1-10-1 MOE 966 398147 48887 48900CTACAGGACAATAC 2-10-2 MOE 957 147706 49133 49144 GCTGACATCTCG 1-10-1 MOE1071 147706 49281 49292 GCTGACATCTCG 1-10-1 MOE 1071 398168 49742 49753TCGGACTTTGAA 1-10-1 MOE 1008 401401 49791 49804 AACTGGGTTAAGTA 2-10-2MOE 958 147689 49936 49947 CAGAGAAGGTCT 1-10-1 MOE 987 401402 5019250205 TGAACACGCTATCC 2-10-2 MOE 959 398117 50241 50254 TTTCCACTTGGGTG2-10-2 MOE 960 147736 50582 50593 AGGTAGGAGAAG 1-10-1 MOE 963 39816850703 50714 TCGGACTTTGAA 1-10-1 MOE 1008 398168 50849 50860 TCGGACTTTGAA1-10-1 MOE 1008 147746 51019 51030 TAAAAACAACAA 1-10-1 MOE 1073 14770851101 51112 TTGATATAGTCA 1-10-1 MOE 997 147746 51178 51189 TAAAAACAACAA1-10-1 MOE 1073 147708 51247 51258 TTGATATAGTCA 1-10-1 MOE 997 14708351281 51292 TACACCAGGTCA 1-10-1 MOE 973 147081 51287 51298 GCTCCTTCCACT1-10-1 MOE 1006 147082 51288 51299 AGCTCCTTCCAC 1-10-1 MOE 1036 14774651331 51342 TAAAAACAACAA 1-10-1 MOE 1073 147085 51416 51427 TCTACACCAGGT1-10-1 MOE 961 147083 51427 51438 TACACCAGGTCA 1-10-1 MOE 973 14708151433 51444 GCTCCTTCCACT 1-10-1 MOE 1006 147082 51434 51445 AGCTCCTTCCAC1-10-1 MOE 1036 147728 51522 51533 GCCAGACAGAAG 1-10-1 MOE 1013 14708551562 51573 TCTACACCAGGT 1-10-1 MOE 961 147081 51633 51644 GCTCCTTCCACT1-10-1 MOE 1006 368360 51633 51646 AAGCTCCTTCCACT 2-10-2 MOE 1035 14708251634 51645 AGCTCCTTCCAC 1-10-1 MOE 1036 368361 51635 51648GAAAGCTCCTTCCA 2-10-2 MOE 962 368360 51779 51792 AAGCTCCTTCCACT 2-10-2MOE 1035 147082 51780 51791 AGCTCCTTCCAC 1-10-1 MOE 1036 147736 5185951870 AGGTAGGAGAAG 1-10-1 MOE 963 147684 51867 51878 ACCCAGTCAGGG 1-10-1MOE 964 147746 51918 51929 TAAAAACAACAA 1-10-1 MOE 1073 147077 5198851999 CTTCCACTGATC 1-10-1 MOE 1047 147746 52064 52075 TAAAAACAACAA1-10-1 MOE 1073 147084 52125 52136 CTACACCAGGTC 1-10-1 MOE 993 14707952136 52147 TCCTTCCACTGA 1-10-1 MOE 1001 147681 52231 52242 ATGTCATTAAAC1-10-1 MOE 965 147084 52271 52282 CTACACCAGGTC 1-10-1 MOE 993 14769152312 52323 GAGGTGGGAAAA 1-10-1 MOE 966 401403 52318 52331TTTCCTAGGAGGTG 2-10-2 MOE 967 398167 52527 52538 CAGGCCATGTGG 1-10-1 MOE1059 147703 52670 52681 TGGCTTCATGTC 1-10-1 MOE 971 398167 52673 52684CAGGCCATGTGG 1-10-1 MOE 1059 398165 52708 52719 GTTCTTAGGAAG 1-10-1 MOE968 398090 52708 52721 TTGTTCTTAGGAAG 2-10-2 MOE 972 147705 52716 52727CGGTTTTTGTTC 1-10-1 MOE 1002 147682 52717 52728 CGGGTACTATGG 1-10-1 MOE992 398167 52762 52773 CAGGCCATGTGG 1-10-1 MOE 1059 147703 52816 52827TGGCTTCATGTC 1-10-1 MOE 971 398090 52854 52867 TTGTTCTTAGGAAG 2-10-2 MOE972 147704 52856 52867 TTGTTCTTAGGA 1-10-1 MOE 1012 147705 52862 52873CGGTTTTTGTTC 1-10-1 MOE 1002 398167 52908 52919 CAGGCCATGTGG 1-10-1 MOE1059 147084 53704 53715 CTACACCAGGTC 1-10-1 MOE 993 147088 53708 53719CCCTCTACACCA 1-10-1 MOE 1050 147083 53849 53860 TACACCAGGTCA 1-10-1 MOE973 147084 53850 53861 CTACACCAGGTC 1-10-1 MOE 993 147086 53852 53863CTCTACACCAGG 1-10-1 MOE 969 147088 53854 53865 CCCTCTACACCA 1-10-1 MOE1050 398167 53870 53881 CAGGCCATGTGG 1-10-1 MOE 1059 147703 54137 54148TGGCTTCATGTC 1-10-1 MOE 971 398155 54172 54185 TGTTTTTACACAGA 2-10-2 MOE970 390030 54263 54274 TTTATAAAACTG 1-10-1 MOE 1074 147705 54275 54286CGGTTTTTGTTC 1-10-1 MOE 1002 147703 54283 54294 TGGCTTCATGTC 1-10-1 MOE971 390030 54409 54420 TTTATAAAACTG 1-10-1 MOE 1074 147704 54965 54976TTGTTCTTAGGA 1-10-1 MOE 1012 147705 54971 54982 CGGTTTTTGTTC 1-10-1 MOE1002 398090 55109 55122 TTGTTCTTAGGAAG 2-10-2 MOE 972 147705 55117 55128CGGTTTTTGTTC 1-10-1 MOE 1002 147083 55206 55217 TACACCAGGTCA 1-10-1 MOE973 147084 55207 55218 CTACACCAGGTC 1-10-1 MOE 993 147084 55353 55364CTACACCAGGTC 1-10-1 MOE 993 147705 55524 55535 CGGTTTTTGTTC 1-10-1 MOE1002 147685 55602 55613 GGCTGACATTCA 1-10-1 MOE 975 401404 55638 55651TGAGCTACAGTAGG 2-10-2 MOE 974 147685 55748 55759 GGCTGACATTCA 1-10-1 MOE975 147712 55819 55830 ACACCATCTCCC 1-10-1 MOE 1005 147712 55965 55976ACACCATCTCCC 1-10-1 MOE 1005 147707 56300 56311 TAGTCATTATCT 1-10-1 MOE977 147708 56306 56317 TTGATATAGTCA 1-10-1 MOE 997 390030 56321 56332TTTATAAAACTG 1-10-1 MOE 1074 147709 56326 56337 CCATTTTTATCA 1-10-1 MOE978 398091 56333 56346 GGGCTTCTTCCATT 2-10-2 MOE 979 401405 56408 56421TGGTCAACTGAAAG 2-10-2 MOE 976 147707 56446 56457 TAGTCATTATCT 1-10-1 MOE977 147708 56452 56463 TTGATATAGTCA 1-10-1 MOE 997 147709 56472 56483CCATTTTTATCA 1-10-1 MOE 978 398091 56479 56492 GGGCTTCTTCCATT 2-10-2 MOE979 401406 56570 56583 GGTGTGGATAACAG 2-10-2 MOE 980 368366 56664 56677CTGATCCTTAGAAG 2-10-2 MOE 1019 398148 57157 57170 TCATAACTATTAAG 2-10-2MOE 981 147082 57220 57231 AGCTCCTTCCAC 1-10-1 MOE 1036 398148 5730357316 TCATAACTATTAAG 2-10-2 MOE 981 147082 57366 57377 AGCTCCTTCCAC1-10-1 MOE 1036 147743 57758 57769 AGGGCTTCCAGT 1-10-1 MOE 1042 39809357963 57976 TCGGACTTTGAAAA 2-10-2 MOE 1009 398093 58109 58122TCGGACTTTGAAAA 2-10-2 MOE 1009 147735 58279 58290 GGAGAAGCGCAG 1-10-1MOE 1016 147087 58821 58832 CCTCTACACCAG 1-10-1 MOE 982 147087 5896758978 CCTCTACACCAG 1-10-1 MOE 982 390030 59180 59191 TTTATAAAACTG 1-10-1MOE 1074 390030 59326 59337 TTTATAAAACTG 1-10-1 MOE 1074 147711 5935759368 AAGGGCCCTGGG 1-10-1 MOE 1040 147743 59382 59393 AGGGCTTCCAGT1-10-1 MOE 1042 147711 59503 59514 AAGGGCCCTGGG 1-10-1 MOE 1040 14771159675 59686 AAGGGCCCTGGG 1-10-1 MOE 1040 401407 59710 59723CAGCTTAGGCAGAG 2-10-2 MOE 983 147712 59711 59722 ACACCATCTCCC 1-10-1 MOE1005 147713 59716 59727 CTCCCACACCAT 1-10-1 MOE 985 147714 59721 59732TTCTGCTCCCAC 1-10-1 MOE 986 147695 59722 59733 TCATTCCCCACT 1-10-1 MOE984 147715 59746 59757 GTTGAGCATGAC 1-10-1 MOE 1077 147711 59821 59832AAGGGCCCTGGG 1-10-1 MOE 1040 390030 59847 59858 TTTATAAAACTG 1-10-1 MOE1074 147712 59857 59868 ACACCATCTCCC 1-10-1 MOE 1005 147713 59862 59873CTCCCACACCAT 1-10-1 MOE 985 147714 59867 59878 TTCTGCTCCCAC 1-10-1 MOE986 390030 59993 60004 TTTATAAAACTG 1-10-1 MOE 1074 389949 60471 60482GCGCGAGCCCGA 1-10-1 MOE 1061 147746 60619 60630 TAAAAACAACAA 1-10-1 MOE1073 147689 61113 61124 CAGAGAAGGTCT 1-10-1 MOE 987 398105 61267 61280TGCACAGGCAGGTT 2-10-2 MOE 1066 147680 61473 61484 GTATGCACTGCT 1-10-1MOE 988 147080 61757 61768 CTCCTTCCACTG 1-10-1 MOE 1021 147078 6190161912 CCTTCCACTGAT 1-10-1 MOE 1044 147079 61902 61913 TCCTTCCACTGA1-10-1 MOE 1001 147088 62215 62226 CCCTCTACACCA 1-10-1 MOE 1050 40140862600 62613 CAATGAAGCACAGG 2-10-2 MOE 989 147688 62843 62854TCCCAAACAAAT 1-10-1 MOE 990 147746 63102 63113 TAAAAACAACAA 1-10-1 MOE1073 147746 63248 63259 TAAAAACAACAA 1-10-1 MOE 1073 401409 63430 63443ATTCTTAACACAGA 2-10-2 MOE 991 147682 63483 63494 CGGGTACTATGG 1-10-1 MOE992 147084 63677 63688 CTACACCAGGTC 1-10-1 MOE 993 147710 64847 64858TATAGCTCCTCT 1-10-1 MOE 994 147710 64993 65004 TATAGCTCCTCT 1-10-1 MOE994 147746 65151 65162 TAAAAACAACAA 1-10-1 MOE 1073 401410 65263 65276CATTTAGGGTCTAA 2-10-2 MOE 995 147717 65862 65873 ATCTTCAGAGAT 1-10-1 MOE996 147717 65895 65906 ATCTTCAGAGAT 1-10-1 MOE 996 147708 65900 65911TTGATATAGTCA 1-10-1 MOE 997 147718 65909 65920 TAATATGACTTG 1-10-1 MOE998 147717 66008 66019 ATCTTCAGAGAT 1-10-1 MOE 996 147717 66041 66052ATCTTCAGAGAT 1-10-1 MOE 996 147708 66046 66057 TTGATATAGTCA 1-10-1 MOE997 147718 66055 66066 TAATATGACTTG 1-10-1 MOE 998 401411 66123 66136AGCCGCCTGAAGTG 2-10-2 MOE 999 147697 66497 66508 CCCCAGCAGCGG 1-10-1 MOE1000 368377 66562 66577 CTCCTTCCACTGA 3-10-3 MOE 1030 TCC 147077 6656366574 CTTCCACTGATC 1-10-1 MOE 1047 368358 66563 66576 TCCTTCCACTGATC2-10-2 MOE 1031 147078 66564 66575 CCTTCCACTGAT 1-10-1 MOE 1044 14707966565 66576 TCCTTCCACTGA 1-10-1 MOE 1001 147080 66566 66577 CTCCTTCCACTG1-10-1 MOE 1021 147697 66643 66654 CCCCAGCAGCGG 1-10-1 MOE 1000 36835866709 66722 TCCTTCCACTGATC 2-10-2 MOE 1031 147078 66710 66721CCTTCCACTGAT 1-10-1 MOE 1044 147079 66711 66722 TCCTTCCACTGA 1-10-1 MOE1001 147075 66999 67010 TCCACTGATCCT 1-10-1 MOE 1026 147705 67067 67078CGGTTTTTGTTC 1-10-1 MOE 1002 147088 67409 67420 CCCTCTACACCA 1-10-1 MOE1050 147080 67430 67441 CTCCTTCCACTG 1-10-1 MOE 1021 147082 67432 67443AGCTCCTTCCAC 1-10-1 MOE 1036 147737 67455 67466 ACAGCCAGGTAG 1-10-1 MOE1067 147088 67555 67566 CCCTCTACACCA 1-10-1 MOE 1050 147082 67578 67589AGCTCCTTCCAC 1-10-1 MOE 1036 401412 67637 67650 TAAATCCTCTAGCA 2-10-2MOE 1003 147091 67729 67740 GTTCCCTCTACA 1-10-1 MOE 1004 147742 6773767748 AACTTCAGTGTC 1-10-1 MOE 1041 147712 68527 68538 ACACCATCTCCC1-10-1 MOE 1005 147712 68673 68684 ACACCATCTCCC 1-10-1 MOE 1005 14771168760 68771 AAGGGCCCTGGG 1-10-1 MOE 1040 147711 68906 68917 AAGGGCCCTGGG1-10-1 MOE 1040 389965 69271 69282 CTGCAACATGAT 1-10-1 MOE 1018 38996569417 69428 CTGCAACATGAT 1-10-1 MOE 1018 368353 69519 69532CACTGATCCTGCAC 2-10-2 MOE 1007 147080 69630 69641 CTCCTTCCACTG 1-10-1MOE 1021 147081 69631 69642 GCTCCTTCCACT 1-10-1 MOE 1006 368353 6966569678 CACTGATCCTGCAC 2-10-2 MOE 1007 398167 69757 69768 CAGGCCATGTGG1-10-1 MOE 1059 398092 69758 69771 AGTCAGGCCATGTG 2-10-2 MOE 1060 39809369811 69824 TCGGACTTTGAAAA 2-10-2 MOE 1009 398168 69813 69824TCGGACTTTGAA 1-10-1 MOE 1008 398167 69903 69914 CAGGCCATGTGG 1-10-1 MOE1059 398093 69957 69970 TCGGACTTTGAAAA 2-10-2 MOE 1009 398094 7004770060 ATCAGCCAGACAGA 2-10-2 MOE 1010 398095 70065 70078 CATCAGCAAGAGGC2-10-2 MOE 1011 147704 70137 70148 TTGTTCTTAGGA 1-10-1 MOE 1012 14772870450 70461 GCCAGACAGAAG 1-10-1 MOE 1013 398164 70464 70475 TTGTCGATCTGC1-10-1 MOE 1014 398096 70562 70575 GGAGAAGCGCAGCT 2-10-2 MOE 1015 14773570564 70575 GGAGAAGCGCAG 1-10-1 MOE 1016 147737 70575 70586 ACAGCCAGGTAG1-10-1 MOE 1067 147735 70710 70721 GGAGAAGCGCAG 1-10-1 MOE 1016 14773770721 70732 ACAGCCAGGTAG 1-10-1 MOE 1067 404131 70729 70742ACCTTCGATCACAG 2-10-2 MOE 831 368349 70762 70775 CTGCACTGACGAGT 2-10-2MOE 1017 389965 70930 70941 CTGCAACATGAT 1-10-1 MOE 1018 368366 7099571008 CTGATCCTTAGAAG 2-10-2 MOE 1019 368354 70999 71012 TCCACTGATCCTGC2-10-2 MOE 1024 368375 71000 71015 CCTTCCACTGATC 3-10-3 MOE 1020 CTG368356 71001 71014 CTTCCACTGATCCT 2-10-2 MOE 1027 368376 71001 71016TCCTTCCACTGAT 3-10-3 MOE 1028 CCT 368357 71002 71015 CCTTCCACTGATCC2-10-2 MOE 1046 368377 71002 71017 CTCCTTCCACTGA 3-10-3 MOE 1030 TCC147077 71003 71014 CTTCCACTGATC 1-10-1 MOE 1047 368358 71003 71016TCCTTCCACTGATC 2-10-2 MOE 1031 368378 71003 71018 GCTCCTTCCACTG 3-10-3MOE 1032 ATC 147078 71004 71015 CCTTCCACTGAT 1-10-1 MOE 1044 36835971005 71018 GCTCCTTCCACTGA 2-10-2 MOE 1033 368379 71005 71020AAGCTCCTTCCAC 3-10-3 MOE 1034 TGA 147080 71006 71017 CTCCTTCCACTG 1-10-1MOE 1021 147082 71008 71019 AGCTCCTTCCAC 1-10-1 MOE 1036 401413 7101971032 TGCAGCCATGTACT 2-10-2 MOE 1022 147738 71067 71078 TGGGTGGCCGGG1-10-1 MOE 1069 147739 71071 71082 CGTTTGGGTGGC 1-10-1 MOE 1023 14774171129 71140 CACCCACTGGTG 1-10-1 MOE 1055 368354 71145 71158TCCACTGATCCTGC 2-10-2 MOE 1024 368355 71146 71159 TTCCACTGATCCTG 2-10-2MOE 1025 147075 71147 71158 TCCACTGATCCT 1-10-1 MOE 1026 368356 7114771160 CTTCCACTGATCCT 2-10-2 MOE 1027 368376 71147 71162 TCCTTCCACTGAT3-10-3 MOE 1028 CCT 147076 71148 71159 TTCCACTGATCC 1-10-1 MOE 1029368357 71148 71161 CCTTCCACTGATCC 2-10-2 MOE 1046 368377 71148 71163CTCCTTCCACTGA 3-10-3 MOE 1030 TCC 147077 71149 71160 CTTCCACTGATC 1-10-1MOE 1047 368358 71149 71162 TCCTTCCACTGATC 2-10-2 MOE 1031 368378 7114971164 GCTCCTTCCACTG 3-10-3 MOE 1032 ATC 147078 71150 71161 CCTTCCACTGAT1-10-1 MOE 1044 368359 71151 71164 GCTCCTTCCACTGA 2-10-2 MOE 1033 36837971151 71166 AAGCTCCTTCCAC 3-10-3 MOE 1034 TGA 368360 71153 71166AAGCTCCTTCCACT 2-10-2 MOE 1035 147082 71154 71165 AGCTCCTTCCAC 1-10-1MOE 1036 368381 71155 71170 GGGAAAGCTCCTT 3-10-3 MOE 1037 CCA 39003071986 71997 TTTATAAAACTG 1-10-1 MOE 1074 390030 72132 72143 TTTATAAAACTG1-10-1 MOE 1074 147711 72300 72311 AAGGGCCCTGGG 1-10-1 MOE 1040 40141472347 72360 TTGCAATGTCTGGC 2-10-2 MOE 1038 147741 72400 72411CACCCACTGGTG 1-10-1 MOE 1055 401415 72415 72428 GATTTATCTGGCTG 2-10-2MOE 1039 147711 72446 72457 AAGGGCCCTGGG 1-10-1 MOE 1040 147742 7257572586 AACTTCAGTGTC 1-10-1 MOE 1041 147743 72690 72701 AGGGCTTCCAGT1-10-1 MOE 1042 147744 72694 72705 AGGAAGGGCTTC 1-10-1 MOE 1043 14774572700 72711 TTGACCAGGAAG 1-10-1 MOE 1058 147742 72721 72732 AACTTCAGTGTC1-10-1 MOE 1041 147743 72836 72847 AGGGCTTCCAGT 1-10-1 MOE 1042 14774472840 72851 AGGAAGGGCTTC 1-10-1 MOE 1043 368357 72898 72911CCTTCCACTGATCC 2-10-2 MOE 1046 147078 72900 72911 CCTTCCACTGAT 1-10-1MOE 1044 398157 72903 72916 GGAAACATACCCTG 2-10-2 MOE 1045 368357 7304473057 CCTTCCACTGATCC 2-10-2 MOE 1046 147077 73045 73056 CTTCCACTGATC1-10-1 MOE 1047 147746 73052 73063 TAAAAACAACAA 1-10-1 MOE 1073 14774673101 73112 TAAAAACAACAA 1-10-1 MOE 1073 398160 73139 73152GAATAGGTTAAGGC 2-10-2 MOE 1048 147746 73198 73209 TAAAAACAACAA 1-10-1MOE 1073 398161 73238 73251 AACAATGTGTTGTA 2-10-2 MOE 1049 147088 7341973430 CCCTCTACACCA 1-10-1 MOE 1050 404140 73457 73470 GCACACAGCTGAGG2-10-2 MOE 1051 404139 73459 73472 GTGCACACAGCTGA 2-10-2 MOE 1052 39930173461 73474 GTGTGCACACAGCT 2-10-2 MOE 1542 404137 73463 73476CAGTGTGCACACAG 2-10-2 MOE 1053 404138 73465 73478 CTCAGTGTGCACAC 2-10-2MOE 1054 147741 73705 73716 CACCCACTGGTG 1-10-1 MOE 1055 404135 7385873871 CATTTCCATGGCCA 2-10-2 MOE 1056 398167 74008 74019 CAGGCCATGTGG1-10-1 MOE 1059 398092 74009 74022 AGTCAGGCCATGTG 2-10-2 MOE 1060 39816274114 74127 ACCAAACAGTTCAG 2-10-2 MOE 1057 147745 74137 74148TTGACCAGGAAG 1-10-1 MOE 1058 398167 74154 74165 CAGGCCATGTGG 1-10-1 MOE1059 398092 74155 74168 AGTCAGGCCATGTG 2-10-2 MOE 1060 389949 7431074321 GCGCGAGCCCGA 1-10-1 MOE 1061 147740 74485 74496 TGTGAGGCTCCA1-10-1 MOE 1062 389950 74527 74538 CCCTGAAGGTTC 1-10-1 MOE 1063 39810174656 74669 TTTGATAAAGCCCT 2-10-2 MOE 1064 398104 74805 74818CAAGAAGACCTTAC 2-10-2 MOE 1065 147737 74893 74904 ACAGCCAGGTAG 1-10-1MOE 1067 398105 74894 74907 TGCACAGGCAGGTT 2-10-2 MOE 1066 147737 7491974930 ACAGCCAGGTAG 1-10-1 MOE 1067 398106 74974 74987 TGGAAAACTGCACC2-10-2 MOE 1068 404199 75045 75058 GGTCATGCACAGGC 2-10-2 MOE 867 40413475048 75061 TCAGGTCATGCACA 2-10-2 MOE 873 398106 75120 75133TGGAAAACTGCACC 2-10-2 MOE 1068 147738 75155 75166 TGGGTGGCCGGG 1-10-1MOE 1069 404132 75227 75240 CCTTGGAATGTCTG 2-10-2 MOE 852 147738 7530175312 TGGGTGGCCGGG 1-10-1 MOE 1069 398166 75499 75510 GGGCTTCTTCCA1-10-1 MOE 1070 147746 75617 75628 TAAAAACAACAA 1-10-1 MOE 1073 14770675686 75697 GCTGACATCTCG 1-10-1 MOE 1071 398112 75730 75743CAGCCTGGCACCTA 2-10-2 MOE 1072 147746 75763 75774 TAAAAACAACAA 1-10-1MOE 1073 398115 75786 75799 AGTAAATATTGGCT 2-10-2 MOE 1076 390030 7583975850 TTTATAAAACTG 1-10-1 MOE 1074 398114 75916 75929 AGGCATATAGCAGA2-10-2 MOE 1075 398115 75932 75945 AGTAAATATTGGCT 2-10-2 MOE 1076 40413375968 75981 TATTCCATGGCCAT 2-10-2 MOE 872 147715 77045 77056GTTGAGCATGAC 1-10-1 MOE 1077 147715 77190 77201 GTTGAGCATGAC 1-10-1 MOE1077 147693 77385 77396 GTGCGCTCCCAT 1-10-1 MOE 1078 398173 40201 40212CAGCCTGGGCAC 1-10-1 MOE 1543 398173 72764 72775 CAGCCTGGGCAC 1-10-1 MOE1543 399096 1986 1999 TGCTCGAACTCCTT 2-10-2 MOE 1544 399102 52822 52835GAAGTCACTGGCTT 2-10-2 MOE 1545 399103 52824 52837 GGGAAGTCACTGGC 2-10-2MOE 1546 399113 59827 59840 GTTAGGCAAAGGGC 2-10-2 MOE 1547 399132 6997769990 GGGCTGAGTGACCC 2-10-2 MOE 1548 399173 74592 74605 ATGCTAGTGCACTA2-10-2 MOE 1549 399208 75900 75913 AGCTCGCTACCTCT 2-10-2 MOE 1550 39927627559 27572 GAGGTATCCCATCT 2-10-2 MOE 1551 399315 74039 74052GGCAACTTCAACCT 2-10-2 MOE 1552

TABLE 19 Short antisense compounds targeted to SEQ ID NO: 12 and having1 or 2 mismatches 5′ 3′ Seq ISIS Target Target Sequence Gapmer ID NO.Site Site (5′-3′) Motif NO 398163 20 31 ATGTCAACCGGC 1-10-1 MOE 908384545 23 34 CAAGTAGGATGT 1-10-1 MOE 951 147733 26 37 TTCTTGATGTCC1-10-1 MOE 891 147721 59 70 AATGCAGGATCT 1-10-1 MOE 1118 147700 110 121GCGCTAGGCCGC 1-10-1 MOE 1110 384545 130 141 CAAGTAGGATGT 1-10-1 MOE 951147705 159 170 CGGTTTTTGTTC 1-10-1 MOE 1002 147701 167 178 CCATGGCGGGAC1-10-1 MOE 921 398164 198 209 TTGTCGATCTGC 1-10-1 MOE 1014 147730 199210 CTTGTCCATCAG 1-10-1 MOE 1121 147702 226 237 CTGGTAAATAGC 1-10-1 MOE898 147703 245 256 TGGCTTCATGTC 1-10-1 MOE 971 147705 266 277CGGTTTTTGTTC 1-10-1 MOE 1002 398165 283 294 GTTCTTAGGAAG 1-10-1 MOE 968147704 285 296 TTGTTCTTAGGA 1-10-1 MOE 1012 147705 291 302 CGGTTTTTGTTC1-10-1 MOE 1002 147709 311 322 CCATTTTTATCA 1-10-1 MOE 978 147733 349360 TTCTTGATGTCC 1-10-1 MOE 891 147707 360 371 TAGTCATTATCT 1-10-1 MOE977 147708 366 377 TTGATATAGTCA 1-10-1 MOE 997 390030 381 392TTTATAAAACTG 1-10-1 MOE 1074 147709 386 397 CCATTTTTATCA 1-10-1 MOE 978147081 393 404 GCTCCTTCCACT 1-10-1 MOE 1006 398091 393 406GGGCTTCTTCCATT 2-10-2 MOE 979 398166 395 406 GGGCTTCTTCCA 1-10-1 MOE1070 147712 461 472 ACACCATCTCCC 1-10-1 MOE 1005 147713 466 477CTCCCACACCAT 1-10-1 MOE 985 147714 471 482 TTCTGCTCCCAC 1-10-1 MOE 986147710 502 513 TATAGCTCCTCT 1-10-1 MOE 994 147736 551 562 AGGTAGGAGAAG1-10-1 MOE 963 147717 574 585 ATCTTCAGAGAT 1-10-1 MOE 996 147717 607 618ATCTTCAGAGAT 1-10-1 MOE 996 147710 609 620 TATAGCTCCTCT 1-10-1 MOE 994147708 612 623 TTGATATAGTCA 1-10-1 MOE 997 147718 621 632 TAATATGACTTG1-10-1 MOE 998 147746 625 636 TAAAAACAACAA 1-10-1 MOE 1073 147736 658669 AGGTAGGAGAAG 1-10-1 MOE 963 147720 676 687 GATCTCTCGAGT 1-10-1 MOE1117 147721 683 694 AATGCAGGATCT 1-10-1 MOE 1118 398167 704 715CAGGCCATGTGG 1-10-1 MOE 1059 398092 705 718 AGTCAGGCCATGTG 2-10-2 MOE1060 147722 709 720 AAAGTCAGGCCA 1-10-1 MOE 1130 147723 715 726GACTCCAAAGTC 1-10-1 MOE 892 147746 733 744 TAAAAACAACAA 1-10-1 MOE 1073398093 758 771 TCGGACTTTGAAAA 2-10-2 MOE 1009 398168 760 771TCGGACTTTGAA 1-10-1 MOE 1008 147725 761 772 CTCGGACTTTGA 1-10-1 MOE 1119147726 766 777 TGACTCTCGGAC 1-10-1 MOE 1120 147738 780 791 TGGGTGGCCGGG1-10-1 MOE 1069 147727 807 818 CAGTGGACCACA 1-10-1 MOE 1128 147728 846857 GCCAGACAGAAG 1-10-1 MOE 1013 398094 848 861 ATCAGCCAGACAGA 2-10-2MOE 1010 398169 849 860 TCAGCCAGACAG 1-10-1 MOE 909 147729 863 874GTAAGAGGCAGG 1-10-1 MOE 920 398095 866 879 CATCAGCAAGAGGC 2-10-2 MOE1011 398164 873 884 TTGTCGATCTGC 1-10-1 MOE 1014 147730 874 885CTTGTCCATCAG 1-10-1 MOE 1121 147731 880 891 TTTCCTCTTGTC 1-10-1 MOE 934147732 885 896 GGGTCTTTCCTC 1-10-1 MOE 1122 147738 888 899 TGGGTGGCCGGG1-10-1 MOE 1069 147733 906 917 TTCTTGATGTCC 1-10-1 MOE 891 398096 971984 GGAGAAGCGCAGCT 2-10-2 MOE 1015 147735 973 984 GGAGAAGCGCAG 1-10-1MOE 1016 147736 978 989 AGGTAGGAGAAG 1-10-1 MOE 963 147729 979 990GTAAGAGGCAGG 1-10-1 MOE 920 147737 984 995 ACAGCCAGGTAG 1-10-1 MOE 1067368349 1025 1038 CTGCACTGACGAGT 2-10-2 MOE 1017 368369 1025 1040TCCTGCACTGACG 3-10-3 MOE 893 AGT 368350 1027 1040 TCCTGCACTGACGA 2-10-2MOE 1079 368370 1027 1042 GATCCTGCACTGA 3-10-3 MOE 1080 CGA 368351 10291042 GATCCTGCACTGAC 2-10-2 MOE 1081 368371 1029 1044 CTGATCCTGCACT3-10-3 MOE 1082 GAC 368352 1031 1044 CTGATCCTGCACTG 2-10-2 MOE 1105368372 1031 1046 CACTGATCCTGCA 3-10-3 MOE 894 CTG 368353 1033 1046CACTGATCCTGCAC 2-10-2 MOE 1007 368373 1033 1048 TCCACTGATCCTG 3-10-3 MOE1083 CAC 368354 1035 1048 TCCACTGATCCTGC 2-10-2 MOE 1024 368368 10351048 TCCACTGATCCTTA 2-10-2 MOE 1127 368374 1035 1050 CTTCCACTGATCC3-10-3 MOE 1126 TGC 368388 1035 1050 CTTCCACTGATCC 3-10-3 MOE 895 TTA147074 1036 1047 CCACTGATCCTG 1-10-1 MOE 845 368355 1036 1049TTCCACTGATCCTG 2-10-2 MOE 1025 368375 1036 1051 CCTTCCACTGATC 3-10-3 MOE1020 CTG 147075 1037 1048 TCCACTGATCCT 1-10-1 MOE 1026 368356 1037 1050CTTCCACTGATCCT 2-10-2 MOE 1027 368376 1037 1052 TCCTTCCACTGAT 3-10-3 MOE1028 CCT 147076 1038 1049 TTCCACTGATCC 1-10-1 MOE 1029 368357 1038 1051CCTTCCACTGATCC 2-10-2 MOE 1046 368377 1038 1053 CTCCTTCCACTGA 3-10-3 MOE1030 TCC 147077 1039 1050 CTTCCACTGATC 1-10-1 MOE 1047 368358 1039 1052TCCTTCCACTGATC 2-10-2 MOE 1031 368378 1039 1054 GCTCCTTCCACTG 3-10-3 MOE1032 ATC 147078 1040 1051 CCTTCCACTGAT 1-10-1 MOE 1044 147079 1041 1052TCCTTCCACTGA 1-10-1 MOE 1001 368359 1041 1054 GCTCCTTCCACTGA 2-10-2 MOE1033 368379 1041 1056 AAGCTCCTTCCAC 3-10-3 MOE 1034 TGA 147080 1042 1053CTCCTTCCACTG 1-10-1 MOE 1021 147081 1043 1054 GCTCCTTCCACT 1-10-1 MOE1006 368360 1043 1056 AAGCTCCTTCCACT 2-10-2 MOE 1035 368380 1043 1058GAAAGCTCCTTCC 3-10-3 MOE 896 ACT 147082 1044 1055 AGCTCCTTCCAC 1-10-1MOE 1036 368361 1045 1058 GAAAGCTCCTTCCA 2-10-2 MOE 962 368381 1045 1060GGGAAAGCTCCTT 3-10-3 MOE 1037 CCA 147729 1087 1098 GTAAGAGGCAGG 1-10-1MOE 920 147738 1103 1114 TGGGTGGCCGGG 1-10-1 MOE 1069 147739 1107 1118CGTTTGGGTGGC 1-10-1 MOE 1023 147740 1124 1135 TGTGAGGCTCCA 1-10-1 MOE1062 398117 1164 1177 TTTCCACTTGGGTG 2-10-2 MOE 960 147741 1165 1176CACCCACTGGTG 1-10-1 MOE 1055 398097 1194 1207 GGCAGTCTTTATCC 2-10-2 MOE897 398098 1272 1285 TAACTTCAGTGTCT 2-10-2 MOE 1131 398117 1272 1285TTTCCACTTGGGTG 2-10-2 MOE 960 147742 1273 1284 AACTTCAGTGTC 1-10-1 MOE1041 147698 1293 1304 CCCGCCACCACC 1-10-1 MOE 928 147743 1388 1399AGGGCTTCCAGT 1-10-1 MOE 1042 398099 1388 1401 GAAGGGCTTCCAGT 2-10-2 MOE1132 147744 1392 1403 AGGAAGGGCTTC 1-10-1 MOE 1043 398100 1395 1408TGACCAGGAAGGGC 2-10-2 MOE 1133 147745 1398 1409 TTGACCAGGAAG 1-10-1 MOE1058 398157 1455 1468 GGAAACATACCCTG 2-10-2 MOE 1045 147745 1458 1469TTGACCAGGAAG 1-10-1 MOE 1058 398167 1475 1486 CAGGCCATGTGG 1-10-1 MOE1059 398118 1564 1577 CGCGAGATATCTAA 2-10-2 MOE 1084 147697 1575 1586CCCCAGCAGCGG 1-10-1 MOE 1000 147076 1596 1607 TTCCACTGATCC 1-10-1 MOE1029 368357 1596 1609 CCTTCCACTGATCC 2-10-2 MOE 1046 147077 1597 1608CTTCCACTGATC 1-10-1 MOE 1047 147078 1598 1609 CCTTCCACTGAT 1-10-1 MOE1044 398118 1672 1685 CGCGAGATATCTAA 2-10-2 MOE 1084 398158 1681 1694AGGCCCTGAGATTA 2-10-2 MOE 1134 147697 1683 1694 CCCCAGCAGCGG 1-10-1 MOE1000 398159 1686 1699 GGTTAAGGCCCTGA 2-10-2 MOE 1135 398160 1691 1704GAATAGGTTAAGGC 2-10-2 MOE 1048 398163 1711 1722 ATGTCAACCGGC 1-10-1 MOE908 147733 1717 1728 TTCTTGATGTCC 1-10-1 MOE 891 147089 1747 1758TCCCTCTACACC 1-10-1 MOE 956 147090 1748 1759 TTCCCTCTACAC 1-10-1 MOE 955147746 1750 1761 TAAAAACAACAA 1-10-1 MOE 1073 389949 1777 1788GCGCGAGCCCGA 1-10-1 MOE 1061 398161 1790 1803 AACAATGTGTTGTA 2-10-2 MOE1049 147746 1799 1810 TAAAAACAACAA 1-10-1 MOE 1073 147700 1801 1812GCGCTAGGCCGC 1-10-1 MOE 1110 147740 1806 1817 TGTGAGGCTCCA 1-10-1 MOE1062 398163 1819 1830 ATGTCAACCGGC 1-10-1 MOE 908 147733 1825 1836TTCTTGATGTCC 1-10-1 MOE 891 389950 1848 1859 CCCTGAAGGTTC 1-10-1 MOE1063 147701 1858 1869 CCATGGCGGGAC 1-10-1 MOE 921 398164 1889 1900TTGTCGATCTGC 1-10-1 MOE 1014 147730 1890 1901 CTTGTCCATCAG 1-10-1 MOE1121 147700 1909 1920 GCGCTAGGCCGC 1-10-1 MOE 1110 398119 1920 1933CGCACCTGGTAAAT 2-10-2 MOE 1085 147685 1957 1968 GGCTGACATTCA 1-10-1 MOE975 147701 1966 1977 CCATGGCGGGAC 1-10-1 MOE 921 398120 1966 1979GTTCAAGCGGCCTA 2-10-2 MOE 1086 398101 1977 1990 TTTGATAAAGCCCT 2-10-2MOE 1064 398164 1997 2008 TTGTCGATCTGC 1-10-1 MOE 1014 147730 1998 2009CTTGTCCATCAG 1-10-1 MOE 1121 147702 2025 2036 CTGGTAAATAGC 1-10-1 MOE898 398119 2028 2041 CGCACCTGGTAAAT 2-10-2 MOE 1085 398120 2074 2087GTTCAAGCGGCCTA 2-10-2 MOE 1086 398105 2099 2112 TGCACAGGCAGGTT 2-10-2MOE 1066 147736 2204 2215 AGGTAGGAGAAG 1-10-1 MOE 963 147741 2257 2268CACCCACTGGTG 1-10-1 MOE 1055 398104 2272 2285 CAAGAAGACCTTAC 2-10-2 MOE1065 147737 2360 2371 ACAGCCAGGTAG 1-10-1 MOE 1067 398105 2361 2374TGCACAGGCAGGTT 2-10-2 MOE 1066 147737 2386 2397 ACAGCCAGGTAG 1-10-1 MOE1067 398095 2407 2420 CATCAGCAAGAGGC 2-10-2 MOE 1011 398106 2441 2454TGGAAAACTGCACC 2-10-2 MOE 1068 398107 2447 2460 TATTCCTGGAAAAC 2-10-2MOE 902 398121 2474 2487 GTGCCTAGCACAGA 2-10-2 MOE 1097 147745 2497 2508TTGACCAGGAAG 1-10-1 MOE 1058 147712 2499 2510 ACACCATCTCCC 1-10-1 MOE1005 398108 2544 2557 GGAATGTCTGAGTT 2-10-2 MOE 1136 147691 2575 2586GAGGTGGGAAAA 1-10-1 MOE 966 398121 2582 2595 GTGCCTAGCACAGA 2-10-2 MOE1097 147738 2622 2633 TGGGTGGCCGGG 1-10-1 MOE 1069 398162 2666 2679ACCAAACAGTTCAG 2-10-2 MOE 1057 147745 2689 2700 TTGACCAGGAAG 1-10-1 MOE1058 398167 2706 2717 CAGGCCATGTGG 1-10-1 MOE 1059 398092 2707 2720AGTCAGGCCATGTG 2-10-2 MOE 1060 398109 2714 2727 CAAGAAGTGTGGTT 2-10-2MOE 903 398110 2852 2865 GTTCCCTTTGCAGG 2-10-2 MOE 952 147091 2854 2865GTTCCCTCTACA 1-10-1 MOE 1004 147723 2924 2935 GACTCCAAAGTC 1-10-1 MOE892 398111 2937 2950 GTGAAAATGCTGGC 2-10-2 MOE 904 398166 2966 2977GGGCTTCTTCCA 1-10-1 MOE 1070 147089 2978 2989 TCCCTCTACACC 1-10-1 MOE956 147090 2979 2990 TTCCCTCTACAC 1-10-1 MOE 955 147706 3007 3018GCTGACATCTCG 1-10-1 MOE 1071 389949 3008 3019 GCGCGAGCCCGA 1-10-1 MOE1061 147723 3032 3043 GACTCCAAAGTC 1-10-1 MOE 892 147740 3037 3048TGTGAGGCTCCA 1-10-1 MOE 1062 398112 3051 3064 CAGCCTGGCACCTA 2-10-2 MOE1072 389950 3079 3090 CCCTGAAGGTTC 1-10-1 MOE 1063 147746 3084 3095TAAAAACAACAA 1-10-1 MOE 1073 398122 3148 3161 CCCTTTACACAAGT 2-10-2 MOE1087 147089 3151 3162 TCCCTCTACACC 1-10-1 MOE 956 147090 3152 3163TTCCCTCTACAC 1-10-1 MOE 955 398113 3160 3173 AGGAGGTTAAACCA 2-10-2 MOE905 147685 3188 3199 GGCTGACATTCA 1-10-1 MOE 975 398101 3208 3221TTTGATAAAGCCCT 2-10-2 MOE 1064 398102 3234 3247 CTACCTGAGGATTT 2-10-2MOE 899 398123 3235 3248 CTCAAAATAGATTT 2-10-2 MOE 1088 398114 3237 3250AGGCATATAGCAGA 2-10-2 MOE 1075 398103 3241 3254 CCCAGTACTACCTG 2-10-2MOE 900 398115 3253 3266 AGTAAATATTGGCT 2-10-2 MOE 1076 398122 3256 3269CCCTTTACACAAGT 2-10-2 MOE 1087 147089 3259 3270 TCCCTCTACACC 1-10-1 MOE956 147090 3260 3271 TTCCCTCTACAC 1-10-1 MOE 955 398116 3266 3279TAATGACCTGATGA 2-10-2 MOE 1137 390030 3306 3317 TTTATAAAACTG 1-10-1 MOE1074 398123 3343 3356 CTCAAAATAGATTT 2-10-2 MOE 1088 147736 3435 3446AGGTAGGAGAAG 1-10-1 MOE 963 398104 3503 3516 CAAGAAGACCTTAC 2-10-2 MOE1065 147737 3591 3602 ACAGCCAGGTAG 1-10-1 MOE 1067 398105 3592 3605TGCACAGGCAGGTT 2-10-2 MOE 1066 147719 3608 3619 CCAACTCCAACT 1-10-1 MOE1116 147737 3617 3628 ACAGCCAGGTAG 1-10-1 MOE 1067 401398 3621 3634CAAAGTCCCTTAGC 2-10-2 MOE 947 147079 3637 3648 TCCTTCCACTGA 1-10-1 MOE1001 147080 3638 3649 CTCCTTCCACTG 1-10-1 MOE 1021 398095 3638 3651CATCAGCAAGAGGC 2-10-2 MOE 1011 398106 3672 3685 TGGAAAACTGCACC 2-10-2MOE 1068 147733 3687 3698 TTCTTGATGTCC 1-10-1 MOE 891 147731 3688 3699TTTCCTCTTGTC 1-10-1 MOE 934 147719 3716 3727 CCAACTCCAACT 1-10-1 MOE1116 147745 3728 3739 TTGACCAGGAAG 1-10-1 MOE 1058 147683 3740 3751GCTTACGATTGT 1-10-1 MOE 922 147079 3745 3756 TCCTTCCACTGA 1-10-1 MOE1001 147080 3746 3757 CTCCTTCCACTG 1-10-1 MOE 1021 398108 3775 3788GGAATGTCTGAGTT 2-10-2 MOE 1136 147733 3795 3806 TTCTTGATGTCC 1-10-1 MOE891 147731 3796 3807 TTTCCTCTTGTC 1-10-1 MOE 934 147691 3806 3817GAGGTGGGAAAA 1-10-1 MOE 966 147738 3853 3864 TGGGTGGCCGGG 1-10-1 MOE1069 398167 3926 3937 CAGGCCATGTGG 1-10-1 MOE 1059 147691 3978 3989GAGGTGGGAAAA 1-10-1 MOE 966 398167 4034 4045 CAGGCCATGTGG 1-10-1 MOE1059 147091 4085 4096 GTTCCCTCTACA 1-10-1 MOE 1004 147691 4086 4097GAGGTGGGAAAA 1-10-1 MOE 966 398111 4168 4181 GTGAAAATGCTGGC 2-10-2 MOE904 398166 4197 4208 GGGCTTCTTCCA 1-10-1 MOE 1070 147091 4223 4234GTTCCCTCTACA 1-10-1 MOE 1004 147092 4224 4235 TGTTCCCTCTAC 1-10-1 MOE901 398112 4282 4295 CAGCCTGGCACCTA 2-10-2 MOE 1072 147746 4315 4326TAAAAACAACAA 1-10-1 MOE 1073 398113 4391 4404 AGGAGGTTAAACCA 2-10-2 MOE905 147723 4422 4433 GACTCCAAAGTC 1-10-1 MOE 892 398114 4468 4481AGGCATATAGCAGA 2-10-2 MOE 1075 398115 4484 4497 AGTAAATATTGGCT 2-10-2MOE 1076 390030 4491 4502 TTTATAAAACTG 1-10-1 MOE 1074 398116 4497 4510TAATGACCTGATGA 2-10-2 MOE 1137 147723 4530 4541 GACTCCAAAGTC 1-10-1 MOE892 390030 4599 4610 TTTATAAAACTG 1-10-1 MOE 1074 398124 4761 4774CACATGAGCTATTC 2-10-2 MOE 1089 398124 4869 4882 CACATGAGCTATTC 2-10-2MOE 1089 147703 4926 4937 TGGCTTCATGTC 1-10-1 MOE 971 147692 4928 4939CTCACCTTCATG 1-10-1 MOE 1113 147696 4975 4986 TGGATGATTGGC 1-10-1 MOE906 147703 5034 5045 TGGCTTCATGTC 1-10-1 MOE 971 147692 5036 5047CTCACCTTCATG 1-10-1 MOE 1113 147098 5173 5184 AGTTGTTGTTCC 1-10-1 MOE1112 398125 5183 5196 CAGTAAGGAATTTT 2-10-2 MOE 913 398126 5216 5229GTGAAGTGAGTCAT 2-10-2 MOE 1090 147098 5281 5292 AGTTGTTGTTCC 1-10-1 MOE1112 398127 5283 5296 GGTCACTCAAGATG 2-10-2 MOE 1091 398126 5324 5337GTGAAGTGAGTCAT 2-10-2 MOE 1090 398128 5335 5348 CTAAATTTAGTTCA 2-10-2MOE 911 398127 5391 5404 GGTCACTCAAGATG 2-10-2 MOE 1091 398128 5443 5456CTAAATTTAGTTCA 2-10-2 MOE 911 147712 5474 5485 ACACCATCTCCC 1-10-1 MOE1005 147736 5600 5611 AGGTAGGAGAAG 1-10-1 MOE 963 147746 5606 5617TAAAAACAACAA 1-10-1 MOE 1073 398129 5628 5641 TTTGAGGAGCTATT 2-10-2 MOE1106 147085 5654 5665 TCTACACCAGGT 1-10-1 MOE 961 147736 5708 5719AGGTAGGAGAAG 1-10-1 MOE 963 398129 5736 5749 TTTGAGGAGCTATT 2-10-2 MOE1106 147679 5934 5945 CAAAAGGATCCC 1-10-1 MOE 907 147723 6229 6240GACTCCAAAGTC 1-10-1 MOE 892 147723 6338 6349 GACTCCAAAGTC 1-10-1 MOE 892390030 6803 6814 TTTATAAAACTG 1-10-1 MOE 1074 398142 6885 6898CCAGCACACTGGAA 2-10-2 MOE 923 390030 6912 6923 TTTATAAAACTG 1-10-1 MOE1074 398142 6994 7007 CCAGCACACTGGAA 2-10-2 MOE 923 147695 7054 7065TCATTCCCCACT 1-10-1 MOE 984 147695 7163 7174 TCATTCCCCACT 1-10-1 MOE 984398166 7197 7208 GGGCTTCTTCCA 1-10-1 MOE 1070 398166 7306 7317GGGCTTCTTCCA 1-10-1 MOE 1070 147684 7442 7453 ACCCAGTCAGGG 1-10-1 MOE964 398130 7694 7707 TTAGTATGACAGCT 2-10-2 MOE 925 398131 7711 7724GGACTCACTCAGCA 2-10-2 MOE 1092 398130 7802 7815 TTAGTATGACAGCT 2-10-2MOE 925 398125 7804 7817 CAGTAAGGAATTTT 2-10-2 MOE 913 398131 7819 7832GGACTCACTCAGCA 2-10-2 MOE 1092 390030 7877 7888 TTTATAAAACTG 1-10-1 MOE1074 398125 7912 7925 CAGTAAGGAATTTT 2-10-2 MOE 913 390030 7985 7996TTTATAAAACTG 1-10-1 MOE 1074 398132 8031 8044 TCAGGGCTACTCAT 2-10-2 MOE1093 398132 8139 8152 TCAGGGCTACTCAT 2-10-2 MOE 1093 147684 8148 8159ACCCAGTCAGGG 1-10-1 MOE 964 147684 8256 8267 ACCCAGTCAGGG 1-10-1 MOE 964398163 8365 8376 ATGTCAACCGGC 1-10-1 MOE 908 398166 8447 8458GGGCTTCTTCCA 1-10-1 MOE 1070 398163 8473 8484 ATGTCAACCGGC 1-10-1 MOE908 398166 8555 8566 GGGCTTCTTCCA 1-10-1 MOE 1070 147718 8631 8642TAATATGACTTG 1-10-1 MOE 998 147691 8698 8709 GAGGTGGGAAAA 1-10-1 MOE 966147691 8806 8817 GAGGTGGGAAAA 1-10-1 MOE 966 147728 8835 8846GCCAGACAGAAG 1-10-1 MOE 1013 147727 8876 8887 CAGTGGACCACA 1-10-1 MOE1128 147728 8943 8954 GCCAGACAGAAG 1-10-1 MOE 1013 398169 8946 8957TCAGCCAGACAG 1-10-1 MOE 909 147727 8984 8995 CAGTGGACCACA 1-10-1 MOE1128 147742 9060 9071 AACTTCAGTGTC 1-10-1 MOE 1041 398133 9112 9125CAGCACTAGATTCA 2-10-2 MOE 1094 384545 9135 9146 CAAGTAGGATGT 1-10-1 MOE951 147742 9168 9179 AACTTCAGTGTC 1-10-1 MOE 1041 398133 9220 9233CAGCACTAGATTCA 2-10-2 MOE 1094 384545 9243 9254 CAAGTAGGATGT 1-10-1 MOE951 398125 9368 9381 CAGTAAGGAATTTT 2-10-2 MOE 913 398125 9476 9489CAGTAAGGAATTTT 2-10-2 MOE 913 401409 9516 9529 ATTCTTAACACAGA 2-10-2 MOE991 147096 9594 9605 TTGTTGTTCCCT 1-10-1 MOE 1107 147733 9597 9608TTCTTGATGTCC 1-10-1 MOE 891 147720 9689 9700 GATCTCTCGAGT 1-10-1 MOE1117 147096 9702 9713 TTGTTGTTCCCT 1-10-1 MOE 1107 147733 9705 9716TTCTTGATGTCC 1-10-1 MOE 891 147720 9797 9808 GATCTCTCGAGT 1-10-1 MOE1117 147746 9963 9974 TAAAAACAACAA 1-10-1 MOE 1073 147746 9966 9977TAAAAACAACAA 1-10-1 MOE 1073 147746 9969 9980 TAAAAACAACAA 1-10-1 MOE1073 147746 9991 10002 TAAAAACAACAA 1-10-1 MOE 1073 147746 10071 10082TAAAAACAACAA 1-10-1 MOE 1073 147746 10074 10085 TAAAAACAACAA 1-10-1 MOE1073 147746 10077 10088 TAAAAACAACAA 1-10-1 MOE 1073 147746 10099 10110TAAAAACAACAA 1-10-1 MOE 1073 398134 10153 10166 TAGCTTAATGTAAC 2-10-2MOE 1095 147085 10221 10232 TCTACACCAGGT 1-10-1 MOE 961 398134 1026110274 TAGCTTAATGTAAC 2-10-2 MOE 1095 390030 10278 10289 TTTATAAAACTG1-10-1 MOE 1074 147084 10328 10339 CTACACCAGGTC 1-10-1 MOE 993 14771110684 10695 AAGGGCCCTGGG 1-10-1 MOE 1040 398128 11333 11346CTAAATTTAGTTCA 2-10-2 MOE 911 398128 11340 11353 CTAAATTTAGTTCA 2-10-2MOE 911 147730 11783 11794 CTTGTCCATCAG 1-10-1 MOE 1121 147731 1178911800 TTTCCTCTTGTC 1-10-1 MOE 934 147730 11790 11801 CTTGTCCATCAG 1-10-1MOE 1121 147731 11796 11807 TTTCCTCTTGTC 1-10-1 MOE 934 147707 1196011971 TAGTCATTATCT 1-10-1 MOE 977 147090 12008 12019 TTCCCTCTACAC 1-10-1MOE 955 147091 12009 12020 GTTCCCTCTACA 1-10-1 MOE 1004 147091 1201412025 GTTCCCTCTACA 1-10-1 MOE 1004 398096 12141 12154 GGAGAAGCGCAGCT2-10-2 MOE 1015 147735 12143 12154 GGAGAAGCGCAG 1-10-1 MOE 1016 39809612146 12159 GGAGAAGCGCAGCT 2-10-2 MOE 1015 147735 12148 12159GGAGAAGCGCAG 1-10-1 MOE 1016 398166 12209 12220 GGGCTTCTTCCA 1-10-1 MOE1070 398166 12214 12225 GGGCTTCTTCCA 1-10-1 MOE 1070 398135 12303 12316GACTACATTTTACA 2-10-2 MOE 912 147741 12389 12400 CACCCACTGGTG 1-10-1 MOE1055 147741 12394 12405 CACCCACTGGTG 1-10-1 MOE 1055 398125 12431 12444CAGTAAGGAATTTT 2-10-2 MOE 913 147714 12585 12596 TTCTGCTCCCAC 1-10-1 MOE986 147718 12594 12605 TAATATGACTTG 1-10-1 MOE 998 398125 12612 12625CAGTAAGGAATTTT 2-10-2 MOE 913 147737 12803 12814 ACAGCCAGGTAG 1-10-1 MOE1067 147746 12876 12887 TAAAAACAACAA 1-10-1 MOE 1073 147691 12900 12911GAGGTGGGAAAA 1-10-1 MOE 966 398136 12915 12928 TTGTGACATCTAGG 2-10-2 MOE1096 147737 12984 12995 ACAGCCAGGTAG 1-10-1 MOE 1067 147746 13057 13068TAAAAACAACAA 1-10-1 MOE 1073 147691 13081 13092 GAGGTGGGAAAA 1-10-1 MOE966 398136 13096 13109 TTGTGACATCTAGG 2-10-2 MOE 1096 398138 13254 13267AACATCAAGCTTGA 2-10-2 MOE 931 398138 13435 13448 AACATCAAGCTTGA 2-10-2MOE 931 147691 13488 13499 GAGGTGGGAAAA 1-10-1 MOE 966 147681 1365913670 ATGTCATTAAAC 1-10-1 MOE 965 147691 13669 13680 GAGGTGGGAAAA 1-10-1MOE 966 389965 13839 13850 CTGCAACATGAT 1-10-1 MOE 1018 389764 1383913850 CTGCAACATGAT 1-9-2 MOE 1018 147681 13840 13851 ATGTCATTAAAC 1-10-1MOE 965 389965 14020 14031 CTGCAACATGAT 1-10-1 MOE 1018 389764 1402014031 CTGCAACATGAT 1-9-2 MOE 1018 389948 14067 14078 CCGTTGGACCCC 1-10-1MOE 915 147736 14123 14134 AGGTAGGAGAAG 1-10-1 MOE 963 389948 1424814259 CCGTTGGACCCC 1-10-1 MOE 915 147738 14279 14290 TGGGTGGCCGGG 1-10-1MOE 1069 147736 14304 14315 AGGTAGGAGAAG 1-10-1 MOE 963 147731 1441114422 TTTCCTCTTGTC 1-10-1 MOE 934 147738 14461 14472 TGGGTGGCCGGG 1-10-1MOE 1069 147692 14475 14486 CTCACCTTCATG 1-10-1 MOE 1113 147731 1459314604 TTTCCTCTTGTC 1-10-1 MOE 934 389950 14614 14625 CCCTGAAGGTTC 1-10-1MOE 1063 147692 14657 14668 CTCACCTTCATG 1-10-1 MOE 1113 147717 1475014761 ATCTTCAGAGAT 1-10-1 MOE 996 147698 14754 14765 CCCGCCACCACC 1-10-1MOE 928 389950 14796 14807 CCCTGAAGGTTC 1-10-1 MOE 1063 398112 1486314876 CAGCCTGGCACCTA 2-10-2 MOE 1072 398121 14875 14888 GTGCCTAGCACAGA2-10-2 MOE 1097 147717 14932 14943 ATCTTCAGAGAT 1-10-1 MOE 996 39811215045 15058 CAGCCTGGCACCTA 2-10-2 MOE 1072 398121 15057 15070GTGCCTAGCACAGA 2-10-2 MOE 1097 147730 15117 15128 CTTGTCCATCAG 1-10-1MOE 1121 147730 15299 15310 CTTGTCCATCAG 1-10-1 MOE 1121 401407 1533915352 CAGCTTAGGCAGAG 2-10-2 MOE 983 398167 15556 15567 CAGGCCATGTGG1-10-1 MOE 1059 147736 16444 16455 AGGTAGGAGAAG 1-10-1 MOE 963 14774616510 16521 TAAAAACAACAA 1-10-1 MOE 1073 147738 16590 16601 TGGGTGGCCGGG1-10-1 MOE 1069 147736 16610 16621 AGGTAGGAGAAG 1-10-1 MOE 963 39816716631 16642 CAGGCCATGTGG 1-10-1 MOE 1059 401411 16657 16670AGCCGCCTGAAGTG 2-10-2 MOE 999 147746 16676 16687 TAAAAACAACAA 1-10-1 MOE1073 398144 16745 16758 GACAGCTTCTATAA 2-10-2 MOE 916 147738 16756 16767TGGGTGGCCGGG 1-10-1 MOE 1069 398167 16797 16808 CAGGCCATGTGG 1-10-1 MOE1059 398144 16911 16924 GACAGCTTCTATAA 2-10-2 MOE 916 389965 17096 17107CTGCAACATGAT 1-10-1 MOE 1018 389764 17096 17107 CTGCAACATGAT 1-9-2 MOE1018 389965 17264 17275 CTGCAACATGAT 1-10-1 MOE 1018 389764 17264 17275CTGCAACATGAT 1-9-2 MOE 1018 147709 17406 17417 CCATTTTTATCA 1-10-1 MOE978 147745 17443 17454 TTGACCAGGAAG 1-10-1 MOE 1058 147746 17497 17508TAAAAACAACAA 1-10-1 MOE 1073 147720 17589 17600 GATCTCTCGAGT 1-10-1 MOE1117 147745 17611 17622 TTGACCAGGAAG 1-10-1 MOE 1058 147695 17634 17645TCATTCCCCACT 1-10-1 MOE 984 147746 17665 17676 TAAAAACAACAA 1-10-1 MOE1073 147088 17707 17718 CCCTCTACACCA 1-10-1 MOE 1050 147720 17757 17768GATCTCTCGAGT 1-10-1 MOE 1117 147711 17808 17819 AAGGGCCCTGGG 1-10-1 MOE1040 147711 17976 17987 AAGGGCCCTGGG 1-10-1 MOE 1040 398139 18049 18062AGTGACTGACCACA 2-10-2 MOE 917 398139 18217 18230 AGTGACTGACCACA 2-10-2MOE 917 398140 18596 18609 GTAGCATAGAGCCT 2-10-2 MOE 918 398140 1876418777 GTAGCATAGAGCCT 2-10-2 MOE 918 398167 18927 18938 CAGGCCATGTGG1-10-1 MOE 1059 398167 19095 19106 CAGGCCATGTGG 1-10-1 MOE 1059 14772419147 19158 GAAATTGAGGAA 1-10-1 MOE 1139 147746 19207 19218 TAAAAACAACAA1-10-1 MOE 1073 147724 19315 19326 GAAATTGAGGAA 1-10-1 MOE 1139 14774019348 19359 TGTGAGGCTCCA 1-10-1 MOE 1062 147746 19375 19386 TAAAAACAACAA1-10-1 MOE 1073 147729 19386 19397 GTAAGAGGCAGG 1-10-1 MOE 920 14770119503 19514 CCATGGCGGGAC 1-10-1 MOE 921 147711 19508 19519 AAGGGCCCTGGG1-10-1 MOE 1040 147740 19516 19527 TGTGAGGCTCCA 1-10-1 MOE 1062 14771819617 19628 TAATATGACTTG 1-10-1 MOE 998 390030 19618 19629 TTTATAAAACTG1-10-1 MOE 1074 147679 19635 19646 CAAAAGGATCCC 1-10-1 MOE 907 14771119676 19687 AAGGGCCCTGGG 1-10-1 MOE 1040 147694 19747 19758 CAGCCTACCAGT1-10-1 MOE 1098 147718 19785 19796 TAATATGACTTG 1-10-1 MOE 998 39003019786 19797 TTTATAAAACTG 1-10-1 MOE 1074 147679 19803 19814 CAAAAGGATCCC1-10-1 MOE 907 147698 19852 19863 CCCGCCACCACC 1-10-1 MOE 928 14769419915 19926 CAGCCTACCAGT 1-10-1 MOE 1098 147704 20011 20022 TTGTTCTTAGGA1-10-1 MOE 1012 147698 20020 20031 CCCGCCACCACC 1-10-1 MOE 928 39814220485 20498 CCAGCACACTGGAA 2-10-2 MOE 923 147078 20514 20525CCTTCCACTGAT 1-10-1 MOE 1044 147079 20515 20526 TCCTTCCACTGA 1-10-1 MOE1001 147080 20516 20527 CTCCTTCCACTG 1-10-1 MOE 1021 398143 20561 20574GTCAGTCCCAGCTA 2-10-2 MOE 924 389965 20620 20631 CTGCAACATGAT 1-10-1 MOE1018 389764 20620 20631 CTGCAACATGAT 1-9-2 MOE 1018 398142 20653 20666CCAGCACACTGGAA 2-10-2 MOE 923 147078 20682 20693 CCTTCCACTGAT 1-10-1 MOE1044 147079 20683 20694 TCCTTCCACTGA 1-10-1 MOE 1001 147080 20684 20695CTCCTTCCACTG 1-10-1 MOE 1021 147080 20704 20715 CTCCTTCCACTG 1-10-1 MOE1021 147081 20705 20716 GCTCCTTCCACT 1-10-1 MOE 1006 398143 20729 20742GTCAGTCCCAGCTA 2-10-2 MOE 924 389965 20788 20799 CTGCAACATGAT 1-10-1 MOE1018 389764 20788 20799 CTGCAACATGAT 1-9-2 MOE 1018 147746 20870 20881TAAAAACAACAA 1-10-1 MOE 1073 147080 20872 20883 CTCCTTCCACTG 1-10-1 MOE1021 147081 20873 20884 GCTCCTTCCACT 1-10-1 MOE 1006 147746 21038 21049TAAAAACAACAA 1-10-1 MOE 1073 147717 21080 21091 ATCTTCAGAGAT 1-10-1 MOE996 147076 21222 21233 TTCCACTGATCC 1-10-1 MOE 1029 147076 21390 21401TTCCACTGATCC 1-10-1 MOE 1029 398094 21441 21454 ATCAGCCAGACAGA 2-10-2MOE 1010 147746 21465 21476 TAAAAACAACAA 1-10-1 MOE 1073 398094 2160921622 ATCAGCCAGACAGA 2-10-2 MOE 1010 398169 21610 21621 TCAGCCAGACAG1-10-1 MOE 909 147746 21633 21644 TAAAAACAACAA 1-10-1 MOE 1073 14773821884 21895 TGGGTGGCCGGG 1-10-1 MOE 1069 147743 22045 22056 AGGGCTTCCAGT1-10-1 MOE 1042 147738 22052 22063 TGGGTGGCCGGG 1-10-1 MOE 1069 14768322107 22118 GCTTACGATTGT 1-10-1 MOE 922 147743 22213 22224 AGGGCTTCCAGT1-10-1 MOE 1042 147681 22566 22577 ATGTCATTAAAC 1-10-1 MOE 965 38995022619 22630 CCCTGAAGGTTC 1-10-1 MOE 1063 147681 22734 22745 ATGTCATTAAAC1-10-1 MOE 965 147736 22759 22770 AGGTAGGAGAAG 1-10-1 MOE 963 38995022787 22798 CCCTGAAGGTTC 1-10-1 MOE 1063 389949 22794 22805 GCGCGAGCCCGA1-10-1 MOE 1061 147736 22927 22938 AGGTAGGAGAAG 1-10-1 MOE 963 38994922962 22973 GCGCGAGCCCGA 1-10-1 MOE 1061 398144 22962 22975GACAGCTTCTATAA 2-10-2 MOE 916 398142 23008 23021 CCAGCACACTGGAA 2-10-2MOE 923 147727 23019 23030 CAGTGGACCACA 1-10-1 MOE 1128 398169 2306423075 TCAGCCAGACAG 1-10-1 MOE 909 398144 23130 23143 GACAGCTTCTATAA2-10-2 MOE 916 398145 23154 23167 ACATGTCAGTAATT 2-10-2 MOE 1099 39814223176 23189 CCAGCACACTGGAA 2-10-2 MOE 923 147727 23187 23198CAGTGGACCACA 1-10-1 MOE 1128 147735 23243 23254 GGAGAAGCGCAG 1-10-1 MOE1016 398145 23322 23335 ACATGTCAGTAATT 2-10-2 MOE 1099 147735 2341123422 GGAGAAGCGCAG 1-10-1 MOE 1016 398146 23478 23491 CTCATGGACACAAA2-10-2 MOE 1100 398146 23646 23659 CTCATGGACACAAA 2-10-2 MOE 1100 39814723784 23797 CTACAGGACAATAC 2-10-2 MOE 957 398114 23853 23866AGGCATATAGCAGA 2-10-2 MOE 1075 398147 23952 23965 CTACAGGACAATAC 2-10-2MOE 957 398114 24021 24034 AGGCATATAGCAGA 2-10-2 MOE 1075 147702 2431924330 CTGGTAAATAGC 1-10-1 MOE 898 147702 24487 24498 CTGGTAAATAGC 1-10-1MOE 898 389965 24543 24554 CTGCAACATGAT 1-10-1 MOE 1018 389764 2454324554 CTGCAACATGAT 1-9-2 MOE 1018 147713 24602 24613 CTCCCACACCAT 1-10-1MOE 985 389965 24711 24722 CTGCAACATGAT 1-10-1 MOE 1018 389764 2471124722 CTGCAACATGAT 1-9-2 MOE 1018 147684 24918 24929 ACCCAGTCAGGG 1-10-1MOE 964 147684 25086 25097 ACCCAGTCAGGG 1-10-1 MOE 964 398148 2515225165 TCATAACTATTAAG 2-10-2 MOE 981 398144 25192 25205 GACAGCTTCTATAA2-10-2 MOE 916 147746 25216 25227 TAAAAACAACAA 1-10-1 MOE 1073 14773625313 25324 AGGTAGGAGAAG 1-10-1 MOE 963 398148 25320 25333TCATAACTATTAAG 2-10-2 MOE 981 398143 25337 25350 GTCAGTCCCAGCTA 2-10-2MOE 924 398144 25360 25373 GACAGCTTCTATAA 2-10-2 MOE 916 147746 2538425395 TAAAAACAACAA 1-10-1 MOE 1073 147691 25442 25453 GAGGTGGGAAAA1-10-1 MOE 966 147736 25481 25492 AGGTAGGAGAAG 1-10-1 MOE 963 39813025504 25517 TTAGTATGACAGCT 2-10-2 MOE 925 147691 25610 25621GAGGTGGGAAAA 1-10-1 MOE 966 147721 25662 25673 AATGCAGGATCT 1-10-1 MOE1118 398130 25672 25685 TTAGTATGACAGCT 2-10-2 MOE 925 147688 25750 25761TCCCAAACAAAT 1-10-1 MOE 990 147746 25810 25821 TAAAAACAACAA 1-10-1 MOE1073 147721 25830 25841 AATGCAGGATCT 1-10-1 MOE 1118 147688 25918 25929TCCCAAACAAAT 1-10-1 MOE 990 147746 25978 25989 TAAAAACAACAA 1-10-1 MOE1073 147746 26172 26183 TAAAAACAACAA 1-10-1 MOE 1073 147746 26340 26351TAAAAACAACAA 1-10-1 MOE 1073 398149 26492 26505 GGAAGTTTTCAAGT 2-10-2MOE 1101 398150 26526 26539 GAATCTGGAGGTAA 2-10-2 MOE 1102 398149 2664126654 GGAAGTTTTCAAGT 2-10-2 MOE 1101 398150 26675 26688 GAATCTGGAGGTAA2-10-2 MOE 1102 147729 26712 26723 GTAAGAGGCAGG 1-10-1 MOE 920 39815126718 26731 TCAGTGTAGGAAGA 2-10-2 MOE 926 147729 26861 26872GTAAGAGGCAGG 1-10-1 MOE 920 398151 26867 26880 TCAGTGTAGGAAGA 2-10-2 MOE926 147728 26917 26928 GCCAGACAGAAG 1-10-1 MOE 1013 147728 27066 27077GCCAGACAGAAG 1-10-1 MOE 1013 147076 27258 27269 TTCCACTGATCC 1-10-1 MOE1029 147731 27267 27278 TTTCCTCTTGTC 1-10-1 MOE 934 147076 27407 27418TTCCACTGATCC 1-10-1 MOE 1029 147731 27416 27427 TTTCCTCTTGTC 1-10-1 MOE934 398152 27559 27572 TGAATATACAGATG 2-10-2 MOE 927 398152 27708 27721TGAATATACAGATG 2-10-2 MOE 927 147696 28265 28276 TGGATGATTGGC 1-10-1 MOE906 147696 28414 28425 TGGATGATTGGC 1-10-1 MOE 906 147698 28481 28492CCCGCCACCACC 1-10-1 MOE 928 147720 28662 28673 GATCTCTCGAGT 1-10-1 MOE1117 389965 28714 28725 CTGCAACATGAT 1-10-1 MOE 1018 389764 28714 28725CTGCAACATGAT 1-9-2 MOE 1018 389965 28861 28872 CTGCAACATGAT 1-10-1 MOE1018 389764 28861 28872 CTGCAACATGAT 1-9-2 MOE 1018 398153 28980 28993ATTTCTCTTACAGG 2-10-2 MOE 948 398153 29126 29139 ATTTCTCTTACAGG 2-10-2MOE 948 147719 29570 29581 CCAACTCCAACT 1-10-1 MOE 1116 398154 2969229705 AGCCCCTTGGCCGT 2-10-2 MOE 1103 147719 29715 29726 CCAACTCCAACT1-10-1 MOE 1116 398155 29785 29798 TGTTTTTACACAGA 2-10-2 MOE 970 39815429837 29850 AGCCCCTTGGCCGT 2-10-2 MOE 1103 401384 29905 29918TGAACACATCACTA 2-10-2 MOE 933 398155 29930 29943 TGTTTTTACACAGA 2-10-2MOE 970 390030 29945 29956 TTTATAAAACTG 1-10-1 MOE 1074 390030 3009030101 TTTATAAAACTG 1-10-1 MOE 1074 398156 30141 30154 GAATACTTCAAATC2-10-2 MOE 1104 398156 30286 30299 GAATACTTCAAATC 2-10-2 MOE 1104 38994830384 30395 CCGTTGGACCCC 1-10-1 MOE 915 389948 30530 30541 CCGTTGGACCCC1-10-1 MOE 915 398142 30591 30604 CCAGCACACTGGAA 2-10-2 MOE 923 14774430654 30665 AGGAAGGGCTTC 1-10-1 MOE 1043 147093 30689 30700 TTGTTCCCTCTA1-10-1 MOE 929 398142 30738 30751 CCAGCACACTGGAA 2-10-2 MOE 923 14774430801 30812 AGGAAGGGCTTC 1-10-1 MOE 1043 398168 31082 31093 TCGGACTTTGAA1-10-1 MOE 1008 147746 31105 31116 TAAAAACAACAA 1-10-1 MOE 1073 39816831230 31241 TCGGACTTTGAA 1-10-1 MOE 1008 390030 31329 31340 TTTATAAAACTG1-10-1 MOE 1074 147736 31458 31469 AGGTAGGAGAAG 1-10-1 MOE 963 39003031477 31488 TTTATAAAACTG 1-10-1 MOE 1074 147736 31606 31617 AGGTAGGAGAAG1-10-1 MOE 963 147698 31713 31724 CCCGCCACCACC 1-10-1 MOE 928 38454531829 31840 CAAGTAGGATGT 1-10-1 MOE 951 147698 31861 31872 CCCGCCACCACC1-10-1 MOE 928 147723 31941 31952 GACTCCAAAGTC 1-10-1 MOE 892 38454531977 31988 CAAGTAGGATGT 1-10-1 MOE 951 147692 32061 32072 CTCACCTTCATG1-10-1 MOE 1113 147723 32089 32100 GACTCCAAAGTC 1-10-1 MOE 892 14769232209 32220 CTCACCTTCATG 1-10-1 MOE 1113 147089 32535 32546 TCCCTCTACACC1-10-1 MOE 956 401396 32569 32582 TGCAGGATGTTGAG 2-10-2 MOE 945 14773032714 32725 CTTGTCCATCAG 1-10-1 MOE 1121 398165 32854 32865 GTTCTTAGGAAG1-10-1 MOE 968 147730 32862 32873 CTTGTCCATCAG 1-10-1 MOE 1121 38995032949 32960 CCCTGAAGGTTC 1-10-1 MOE 1063 398165 33002 33013 GTTCTTAGGAAG1-10-1 MOE 968 147736 33012 33023 AGGTAGGAGAAG 1-10-1 MOE 963 36835233056 33069 CTGATCCTGCACTG 2-10-2 MOE 1105 147081 33073 33084GCTCCTTCCACT 1-10-1 MOE 1006 368360 33073 33086 AAGCTCCTTCCACT 2-10-2MOE 1035 147082 33074 33085 AGCTCCTTCCAC 1-10-1 MOE 1036 389950 3309733108 CCCTGAAGGTTC 1-10-1 MOE 1063 147736 33160 33171 AGGTAGGAGAAG1-10-1 MOE 963 368352 33204 33217 CTGATCCTGCACTG 2-10-2 MOE 1105 14708133221 33232 GCTCCTTCCACT 1-10-1 MOE 1006 147082 33222 33233 AGCTCCTTCCAC1-10-1 MOE 1036 398138 33244 33257 AACATCAAGCTTGA 2-10-2 MOE 931 14774633250 33261 TAAAAACAACAA 1-10-1 MOE 1073 398138 33392 33405AACATCAAGCTTGA 2-10-2 MOE 931 147746 33398 33409 TAAAAACAACAA 1-10-1 MOE1073 147732 33652 33663 GGGTCTTTCCTC 1-10-1 MOE 1122 147724 33733 33744GAAATTGAGGAA 1-10-1 MOE 1139 147732 33800 33811 GGGTCTTTCCTC 1-10-1 MOE1122 147724 33881 33892 GAAATTGAGGAA 1-10-1 MOE 1139 147719 33976 33987CCAACTCCAACT 1-10-1 MOE 1116 147746 34034 34045 TAAAAACAACAA 1-10-1 MOE1073 398129 34045 34058 TTTGAGGAGCTATT 2-10-2 MOE 1106 147719 3412434135 CCAACTCCAACT 1-10-1 MOE 1116 147721 34156 34167 AATGCAGGATCT1-10-1 MOE 1118 398129 34193 34206 TTTGAGGAGCTATT 2-10-2 MOE 1106 14772134304 34315 AATGCAGGATCT 1-10-1 MOE 1118 147746 34606 34617 TAAAAACAACAA1-10-1 MOE 1073 398165 34704 34715 GTTCTTAGGAAG 1-10-1 MOE 968 14774634754 34765 TAAAAACAACAA 1-10-1 MOE 1073 398165 34852 34863 GTTCTTAGGAAG1-10-1 MOE 968 147717 34893 34904 ATCTTCAGAGAT 1-10-1 MOE 996 14771934976 34987 CCAACTCCAACT 1-10-1 MOE 1116 147092 34987 34998 TGTTCCCTCTAC1-10-1 MOE 901 147719 35124 35135 CCAACTCCAACT 1-10-1 MOE 1116 14709235135 35146 TGTTCCCTCTAC 1-10-1 MOE 901 147736 35248 35259 AGGTAGGAGAAG1-10-1 MOE 963 147738 35391 35402 TGGGTGGCCGGG 1-10-1 MOE 1069 14773635396 35407 AGGTAGGAGAAG 1-10-1 MOE 963 147738 35539 35550 TGGGTGGCCGGG1-10-1 MOE 1069 147691 35554 35565 GAGGTGGGAAAA 1-10-1 MOE 966 14769135702 35713 GAGGTGGGAAAA 1-10-1 MOE 966 147746 35814 35825 TAAAAACAACAA1-10-1 MOE 1073 147733 35889 35900 TTCTTGATGTCC 1-10-1 MOE 891 14773335923 35934 TTCTTGATGTCC 1-10-1 MOE 891 147746 35962 35973 TAAAAACAACAA1-10-1 MOE 1073 147726 35978 35989 TGACTCTCGGAC 1-10-1 MOE 1120 14773336037 36048 TTCTTGATGTCC 1-10-1 MOE 891 147733 36071 36082 TTCTTGATGTCC1-10-1 MOE 891 147726 36126 36137 TGACTCTCGGAC 1-10-1 MOE 1120 14773636359 36370 AGGTAGGAGAAG 1-10-1 MOE 963 147691 36360 36371 GAGGTGGGAAAA1-10-1 MOE 966 147736 36507 36518 AGGTAGGAGAAG 1-10-1 MOE 963 14769136508 36519 GAGGTGGGAAAA 1-10-1 MOE 966 147746 36564 36575 TAAAAACAACAA1-10-1 MOE 1073 147723 36575 36586 GACTCCAAAGTC 1-10-1 MOE 892 14773136620 36631 TTTCCTCTTGTC 1-10-1 MOE 934 147723 36723 36734 GACTCCAAAGTC1-10-1 MOE 892 147731 36768 36779 TTTCCTCTTGTC 1-10-1 MOE 934 39816937174 37185 TCAGCCAGACAG 1-10-1 MOE 909 147688 37380 37391 TCCCAAACAAAT1-10-1 MOE 990 147688 37528 37539 TCCCAAACAAAT 1-10-1 MOE 990 14771437881 37892 TTCTGCTCCCAC 1-10-1 MOE 986 147714 38029 38040 TTCTGCTCCCAC1-10-1 MOE 986 147681 38364 38375 ATGTCATTAAAC 1-10-1 MOE 965 14773638766 38777 AGGTAGGAGAAG 1-10-1 MOE 963 147738 38909 38920 TGGGTGGCCGGG1-10-1 MOE 1069 147736 38914 38925 AGGTAGGAGAAG 1-10-1 MOE 963 14773839057 39068 TGGGTGGCCGGG 1-10-1 MOE 1069 390030 39249 39260 TTTATAAAACTG1-10-1 MOE 1074 390030 39397 39408 TTTATAAAACTG 1-10-1 MOE 1074 14771739545 39556 ATCTTCAGAGAT 1-10-1 MOE 996 147717 39693 39704 ATCTTCAGAGAT1-10-1 MOE 996 147746 39729 39740 TAAAAACAACAA 1-10-1 MOE 1073 14774639789 39800 TAAAAACAACAA 1-10-1 MOE 1073 147691 39829 39840 GAGGTGGGAAAA1-10-1 MOE 966 147746 39877 39888 TAAAAACAACAA 1-10-1 MOE 1073 14769139977 39988 GAGGTGGGAAAA 1-10-1 MOE 966 147727 39983 39994 CAGTGGACCACA1-10-1 MOE 1128 147727 40131 40142 CAGTGGACCACA 1-10-1 MOE 1128 14774640333 40344 TAAAAACAACAA 1-10-1 MOE 1073 147719 40457 40468 CCAACTCCAACT1-10-1 MOE 1116 147679 40467 40478 CAAAAGGATCCC 1-10-1 MOE 907 14774640478 40489 TAAAAACAACAA 1-10-1 MOE 1073 147741 40565 40576 CACCCACTGGTG1-10-1 MOE 1055 398166 40589 40600 GGGCTTCTTCCA 1-10-1 MOE 1070 14771940605 40616 CCAACTCCAACT 1-10-1 MOE 1116 147679 40615 40626 CAAAAGGATCCC1-10-1 MOE 907 147746 40626 40637 TAAAAACAACAA 1-10-1 MOE 1073 14773540662 40673 GGAGAAGCGCAG 1-10-1 MOE 1016 147746 40706 40717 TAAAAACAACAA1-10-1 MOE 1073 147741 40713 40724 CACCCACTGGTG 1-10-1 MOE 1055 39816640737 40748 GGGCTTCTTCCA 1-10-1 MOE 1070 147735 40810 40821 GGAGAAGCGCAG1-10-1 MOE 1016 147746 40854 40865 TAAAAACAACAA 1-10-1 MOE 1073 14771841218 41229 TAATATGACTTG 1-10-1 MOE 998 147717 41221 41232 ATCTTCAGAGAT1-10-1 MOE 996 147717 41369 41380 ATCTTCAGAGAT 1-10-1 MOE 996 14772341627 41638 GACTCCAAAGTC 1-10-1 MOE 892 147717 41747 41758 ATCTTCAGAGAT1-10-1 MOE 996 147723 41775 41786 GACTCCAAAGTC 1-10-1 MOE 892 39003041908 41919 TTTATAAAACTG 1-10-1 MOE 1074 390030 42056 42067 TTTATAAAACTG1-10-1 MOE 1074 398153 42157 42170 ATTTCTCTTACAGG 2-10-2 MOE 948 39815342305 42318 ATTTCTCTTACAGG 2-10-2 MOE 948 147690 42423 42434TGAAGTTAATTC 1-10-1 MOE 1138 147695 42521 42532 TCATTCCCCACT 1-10-1 MOE984 147710 42543 42554 TATAGCTCCTCT 1-10-1 MOE 994 147690 42571 42582TGAAGTTAATTC 1-10-1 MOE 1138 147695 42669 42680 TCATTCCCCACT 1-10-1 MOE984 147078 43321 43332 CCTTCCACTGAT 1-10-1 MOE 1044 147079 43322 43333TCCTTCCACTGA 1-10-1 MOE 1001 147716 43329 43340 TTAACGAGCCTT 1-10-1 MOE949 147078 43469 43480 CCTTCCACTGAT 1-10-1 MOE 1044 147079 43470 43481TCCTTCCACTGA 1-10-1 MOE 1001 147080 43471 43482 CTCCTTCCACTG 1-10-1 MOE1021 398102 43837 43850 CTACCTGAGGATTT 2-10-2 MOE 899 147074 43848 43859CCACTGATCCTG 1-10-1 MOE 845 401408 43871 43884 CAATGAAGCACAGG 2-10-2 MOE989 398102 43985 43998 CTACCTGAGGATTT 2-10-2 MOE 899 147736 44137 44148AGGTAGGAGAAG 1-10-1 MOE 963 147746 44140 44151 TAAAAACAACAA 1-10-1 MOE1073 147687 44206 44217 CGACACGGGAAC 1-10-1 MOE 950 147743 44223 44234AGGGCTTCCAGT 1-10-1 MOE 1042 384545 44242 44253 CAAGTAGGATGT 1-10-1 MOE951 147736 44285 44296 AGGTAGGAGAAG 1-10-1 MOE 963 147743 44371 44382AGGGCTTCCAGT 1-10-1 MOE 1042 384545 44390 44401 CAAGTAGGATGT 1-10-1 MOE951 147728 44589 44600 GCCAGACAGAAG 1-10-1 MOE 1013 389948 44628 44639CCGTTGGACCCC 1-10-1 MOE 915 147720 44703 44714 GATCTCTCGAGT 1-10-1 MOE1117 147728 44729 44740 GCCAGACAGAAG 1-10-1 MOE 1013 147728 44737 44748GCCAGACAGAAG 1-10-1 MOE 1013 389948 44776 44787 CCGTTGGACCCC 1-10-1 MOE915 147720 44851 44862 GATCTCTCGAGT 1-10-1 MOE 1117 398110 44861 44874GTTCCCTTTGCAGG 2-10-2 MOE 952 147728 44877 44888 GCCAGACAGAAG 1-10-1 MOE1013 147705 45092 45103 CGGTTTTTGTTC 1-10-1 MOE 1002 147705 45240 45251CGGTTTTTGTTC 1-10-1 MOE 1002 147681 45337 45348 ATGTCATTAAAC 1-10-1 MOE965 147681 45485 45496 ATGTCATTAAAC 1-10-1 MOE 965 147096 45660 45671TTGTTGTTCCCT 1-10-1 MOE 1107 147096 45808 45819 TTGTTGTTCCCT 1-10-1 MOE1107 368368 45976 45989 TCCACTGATCCTTA 2-10-2 MOE 1127 147074 4597745988 CCACTGATCCTG 1-10-1 MOE 845 147075 45978 45989 TCCACTGATCCT 1-10-1MOE 1026 147076 45979 45990 TTCCACTGATCC 1-10-1 MOE 1029 368368 4612446137 TCCACTGATCCTTA 2-10-2 MOE 1127 147075 46126 46137 TCCACTGATCCT1-10-1 MOE 1026 147076 46127 46138 TTCCACTGATCC 1-10-1 MOE 1029 14770546555 46566 CGGTTTTTGTTC 1-10-1 MOE 1002 147714 46685 46696 TTCTGCTCCCAC1-10-1 MOE 986 147705 46703 46714 CGGTTTTTGTTC 1-10-1 MOE 1002 14771446833 46844 TTCTGCTCCCAC 1-10-1 MOE 986 390030 47007 47018 TTTATAAAACTG1-10-1 MOE 1074 147746 47023 47034 TAAAAACAACAA 1-10-1 MOE 1073 14774647171 47182 TAAAAACAACAA 1-10-1 MOE 1073 147085 47607 47618 TCTACACCAGGT1-10-1 MOE 961 147746 47609 47620 TAAAAACAACAA 1-10-1 MOE 1073 14708947611 47622 TCCCTCTACACC 1-10-1 MOE 956 147091 47613 47624 GTTCCCTCTACA1-10-1 MOE 1004 401384 47689 47702 TGAACACATCACTA 2-10-2 MOE 933 14769147729 47740 GAGGTGGGAAAA 1-10-1 MOE 966 147085 47755 47766 TCTACACCAGGT1-10-1 MOE 961 147087 47757 47768 CCTCTACACCAG 1-10-1 MOE 982 14709047760 47771 TTCCCTCTACAC 1-10-1 MOE 955 147091 47761 47772 GTTCCCTCTACA1-10-1 MOE 1004 147099 47770 47781 GAGTTGTTGTTC 1-10-1 MOE 1108 14710047771 47782 CGAGTTGTTGTT 1-10-1 MOE 1109 390030 47847 47858 TTTATAAAACTG1-10-1 MOE 1074 147691 47877 47888 GAGGTGGGAAAA 1-10-1 MOE 966 14709947918 47929 GAGTTGTTGTTC 1-10-1 MOE 1108 147100 47919 47930 CGAGTTGTTGTT1-10-1 MOE 1109 390030 47995 48006 TTTATAAAACTG 1-10-1 MOE 1074 14707448222 48233 CCACTGATCCTG 1-10-1 MOE 845 147731 48340 48351 TTTCCTCTTGTC1-10-1 MOE 934 147691 48393 48404 GAGGTGGGAAAA 1-10-1 MOE 966 14773148488 48499 TTTCCTCTTGTC 1-10-1 MOE 934 147691 48541 48552 GAGGTGGGAAAA1-10-1 MOE 966 398147 48887 48900 CTACAGGACAATAC 2-10-2 MOE 957 39814749035 49048 CTACAGGACAATAC 2-10-2 MOE 957 147074 49525 49536CCACTGATCCTG 1-10-1 MOE 845 398168 49742 49753 TCGGACTTTGAA 1-10-1 MOE1008 384545 49858 49869 CAAGTAGGATGT 1-10-1 MOE 951 398168 49890 49901TCGGACTTTGAA 1-10-1 MOE 1008 147724 49974 49985 GAAATTGAGGAA 1-10-1 MOE1139 384545 50006 50017 CAAGTAGGATGT 1-10-1 MOE 951 147689 50084 50095CAGAGAAGGTCT 1-10-1 MOE 987 147687 50102 50113 CGACACGGGAAC 1-10-1 MOE950 147724 50122 50133 GAAATTGAGGAA 1-10-1 MOE 1139 147687 50250 50261CGACACGGGAAC 1-10-1 MOE 950 398117 50389 50402 TTTCCACTTGGGTG 2-10-2 MOE960 147736 50436 50447 AGGTAGGAGAAG 1-10-1 MOE 963 147736 50582 50593AGGTAGGAGAAG 1-10-1 MOE 963 398168 50703 50714 TCGGACTTTGAA 1-10-1 MOE1008 401397 50822 50835 CTGGTCAGCATTGA 2-10-2 MOE 946 147746 51019 51030TAAAAACAACAA 1-10-1 MOE 1073 147708 51101 51112 TTGATATAGTCA 1-10-1 MOE997 147746 51165 51176 TAAAAACAACAA 1-10-1 MOE 1073 147746 51185 51196TAAAAACAACAA 1-10-1 MOE 1073 147708 51247 51258 TTGATATAGTCA 1-10-1 MOE997 147081 51287 51298 GCTCCTTCCACT 1-10-1 MOE 1006 147082 51288 51299AGCTCCTTCCAC 1-10-1 MOE 1036 147746 51324 51335 TAAAAACAACAA 1-10-1 MOE1073 147746 51331 51342 TAAAAACAACAA 1-10-1 MOE 1073 147728 51376 51387GCCAGACAGAAG 1-10-1 MOE 1013 147729 51406 51417 GTAAGAGGCAGG 1-10-1 MOE920 147081 51433 51444 GCTCCTTCCACT 1-10-1 MOE 1006 147082 51434 51445AGCTCCTTCCAC 1-10-1 MOE 1036 147728 51492 51503 GCCAGACAGAAG 1-10-1 MOE1013 147728 51522 51533 GCCAGACAGAAG 1-10-1 MOE 1013 147729 51552 51563GTAAGAGGCAGG 1-10-1 MOE 920 368360 51633 51646 AAGCTCCTTCCACT 2-10-2 MOE1035 147082 51634 51645 AGCTCCTTCCAC 1-10-1 MOE 1036 368361 51635 51648GAAAGCTCCTTCCA 2-10-2 MOE 962 147728 51638 51649 GCCAGACAGAAG 1-10-1 MOE1013 147695 51644 51655 TCATTCCCCACT 1-10-1 MOE 984 147736 51713 51724AGGTAGGAGAAG 1-10-1 MOE 963 147684 51721 51732 ACCCAGTCAGGG 1-10-1 MOE964 147081 51779 51790 GCTCCTTCCACT 1-10-1 MOE 1006 368360 51779 51792AAGCTCCTTCCACT 2-10-2 MOE 1035 147082 51780 51791 AGCTCCTTCCAC 1-10-1MOE 1036 368361 51781 51794 GAAAGCTCCTTCCA 2-10-2 MOE 962 147695 5179051801 TCATTCCCCACT 1-10-1 MOE 984 147736 51859 51870 AGGTAGGAGAAG 1-10-1MOE 963 147077 51988 51999 CTTCCACTGATC 1-10-1 MOE 1047 147079 5199052001 TCCTTCCACTGA 1-10-1 MOE 1001 147746 52064 52075 TAAAAACAACAA1-10-1 MOE 1073 147681 52085 52096 ATGTCATTAAAC 1-10-1 MOE 965 14707752134 52145 CTTCCACTGATC 1-10-1 MOE 1047 147079 52136 52147 TCCTTCCACTGA1-10-1 MOE 1001 147691 52166 52177 GAGGTGGGAAAA 1-10-1 MOE 966 14771952252 52263 CCAACTCCAACT 1-10-1 MOE 1116 147691 52312 52323 GAGGTGGGAAAA1-10-1 MOE 966 147719 52398 52409 CCAACTCCAACT 1-10-1 MOE 1116 14772852428 52439 GCCAGACAGAAG 1-10-1 MOE 1013 147729 52483 52494 GTAAGAGGCAGG1-10-1 MOE 920 398167 52527 52538 CAGGCCATGTGG 1-10-1 MOE 1059 14768252571 52582 CGGGTACTATGG 1-10-1 MOE 992 147728 52574 52585 GCCAGACAGAAG1-10-1 MOE 1013 147724 52615 52626 GAAATTGAGGAA 1-10-1 MOE 1139 14772952629 52640 GTAAGAGGCAGG 1-10-1 MOE 920 147703 52670 52681 TGGCTTCATGTC1-10-1 MOE 971 398167 52673 52684 CAGGCCATGTGG 1-10-1 MOE 1059 39816552708 52719 GTTCTTAGGAAG 1-10-1 MOE 968 147704 52710 52721 TTGTTCTTAGGA1-10-1 MOE 1012 147705 52716 52727 CGGTTTTTGTTC 1-10-1 MOE 1002 14772452761 52772 GAAATTGAGGAA 1-10-1 MOE 1139 398167 52762 52773 CAGGCCATGTGG1-10-1 MOE 1059 147703 52816 52827 TGGCTTCATGTC 1-10-1 MOE 971 39816552854 52865 GTTCTTAGGAAG 1-10-1 MOE 968 147704 52856 52867 TTGTTCTTAGGA1-10-1 MOE 1012 147705 52862 52873 CGGTTTTTGTTC 1-10-1 MOE 1002 39816752908 52919 CAGGCCATGTGG 1-10-1 MOE 1059 147689 53063 53074 CAGAGAAGGTCT1-10-1 MOE 987 147727 53111 53122 CAGTGGACCACA 1-10-1 MOE 1128 14772753158 53169 CAGTGGACCACA 1-10-1 MOE 1128 147689 53209 53220 CAGAGAAGGTCT1-10-1 MOE 987 147727 53257 53268 CAGTGGACCACA 1-10-1 MOE 1128 14772753304 53315 CAGTGGACCACA 1-10-1 MOE 1128 147680 53638 53649 GTATGCACTGCT1-10-1 MOE 988 147722 53650 53661 AAAGTCAGGCCA 1-10-1 MOE 1130 14708353703 53714 TACACCAGGTCA 1-10-1 MOE 973 147085 53705 53716 TCTACACCAGGT1-10-1 MOE 961 147086 53706 53717 CTCTACACCAGG 1-10-1 MOE 969 39816753724 53735 CAGGCCATGTGG 1-10-1 MOE 1059 147684 53747 53758 ACCCAGTCAGGG1-10-1 MOE 964 147680 53784 53795 GTATGCACTGCT 1-10-1 MOE 988 14772253796 53807 AAAGTCAGGCCA 1-10-1 MOE 1130 147085 53851 53862 TCTACACCAGGT1-10-1 MOE 961 398167 53870 53881 CAGGCCATGTGG 1-10-1 MOE 1059 14768453893 53904 ACCCAGTCAGGG 1-10-1 MOE 964 398155 54026 54039TGTTTTTACACAGA 2-10-2 MOE 970 147703 54137 54148 TGGCTTCATGTC 1-10-1 MOE971 398155 54172 54185 TGTTTTTACACAGA 2-10-2 MOE 970 147705 54275 54286CGGTTTTTGTTC 1-10-1 MOE 1002 147703 54283 54294 TGGCTTCATGTC 1-10-1 MOE971 147705 54421 54432 CGGTTTTTGTTC 1-10-1 MOE 1002 147727 54853 54864CAGTGGACCACA 1-10-1 MOE 1128 398165 54963 54974 GTTCTTAGGAAG 1-10-1 MOE968 398090 54963 54976 TTGTTCTTAGGAAG 2-10-2 MOE 972 147704 54965 54976TTGTTCTTAGGA 1-10-1 MOE 1012 147705 54971 54982 CGGTTTTTGTTC 1-10-1 MOE1002 147727 54999 55010 CAGTGGACCACA 1-10-1 MOE 1128 398165 55109 55120GTTCTTAGGAAG 1-10-1 MOE 968 147704 55111 55122 TTGTTCTTAGGA 1-10-1 MOE1012 147705 55117 55128 CGGTTTTTGTTC 1-10-1 MOE 1002 147083 55352 55363TACACCAGGTCA 1-10-1 MOE 973 147705 55378 55389 CGGTTTTTGTTC 1-10-1 MOE1002 147705 55524 55535 CGGTTTTTGTTC 1-10-1 MOE 1002 147712 55819 55830ACACCATCTCCC 1-10-1 MOE 1005 147712 55965 55976 ACACCATCTCCC 1-10-1 MOE1005 147733 56289 56300 TTCTTGATGTCC 1-10-1 MOE 891 147707 56300 56311TAGTCATTATCT 1-10-1 MOE 977 147708 56306 56317 TTGATATAGTCA 1-10-1 MOE997 390030 56321 56332 TTTATAAAACTG 1-10-1 MOE 1074 147081 56333 56344GCTCCTTCCACT 1-10-1 MOE 1006 398166 56335 56346 GGGCTTCTTCCA 1-10-1 MOE1070 147733 56435 56446 TTCTTGATGTCC 1-10-1 MOE 891 147707 56446 56457TAGTCATTATCT 1-10-1 MOE 977 147708 56452 56463 TTGATATAGTCA 1-10-1 MOE997 390030 56467 56478 TTTATAAAACTG 1-10-1 MOE 1074 147081 56479 56490GCTCCTTCCACT 1-10-1 MOE 1006 398091 56479 56492 GGGCTTCTTCCATT 2-10-2MOE 979 398166 56481 56492 GGGCTTCTTCCA 1-10-1 MOE 1070 368366 5651856531 CTGATCCTTAGAAG 2-10-2 MOE 1019 147743 57612 57623 AGGGCTTCCAGT1-10-1 MOE 1042 147700 57709 57720 GCGCTAGGCCGC 1-10-1 MOE 1110 14774357758 57769 AGGGCTTCCAGT 1-10-1 MOE 1042 147700 57855 57866 GCGCTAGGCCGC1-10-1 MOE 1110 398093 57963 57976 TCGGACTTTGAAAA 2-10-2 MOE 1009 39816857965 57976 TCGGACTTTGAA 1-10-1 MOE 1008 147698 58105 58116 CCCGCCACCACC1-10-1 MOE 928 398093 58109 58122 TCGGACTTTGAAAA 2-10-2 MOE 1009 39816858111 58122 TCGGACTTTGAA 1-10-1 MOE 1008 147698 58251 58262 CCCGCCACCACC1-10-1 MOE 928 147735 58279 58290 GGAGAAGCGCAG 1-10-1 MOE 1016 14773558425 58436 GGAGAAGCGCAG 1-10-1 MOE 1016 404135 58946 58959CATTTCCATGGCCA 2-10-2 MOE 1056 390030 59326 59337 TTTATAAAACTG 1-10-1MOE 1074 147711 59357 59368 AAGGGCCCTGGG 1-10-1 MOE 1040 147743 5938259393 AGGGCTTCCAGT 1-10-1 MOE 1042 147711 59503 59514 AAGGGCCCTGGG1-10-1 MOE 1040 147743 59528 59539 AGGGCTTCCAGT 1-10-1 MOE 1042 14769559576 59587 TCATTCCCCACT 1-10-1 MOE 984 147713 59716 59727 CTCCCACACCAT1-10-1 MOE 985 147714 59721 59732 TTCTGCTCCCAC 1-10-1 MOE 986 14771559746 59757 GTTGAGCATGAC 1-10-1 MOE 1077 147716 59771 59782 TTAACGAGCCTT1-10-1 MOE 949 147712 59857 59868 ACACCATCTCCC 1-10-1 MOE 1005 14771459867 59878 TTCTGCTCCCAC 1-10-1 MOE 986 147715 59892 59903 GTTGAGCATGAC1-10-1 MOE 1077 147716 59917 59928 TTAACGAGCCTT 1-10-1 MOE 949 39003059993 60004 TTTATAAAACTG 1-10-1 MOE 1074 147690 60270 60281 TGAAGTTAATTC1-10-1 MOE 1138 389949 60325 60336 GCGCGAGCCCGA 1-10-1 MOE 1061 14769060416 60427 TGAAGTTAATTC 1-10-1 MOE 1138 389949 60471 60482 GCGCGAGCCCGA1-10-1 MOE 1061 147746 60619 60630 TAAAAACAACAA 1-10-1 MOE 1073 38454560676 60687 CAAGTAGGATGT 1-10-1 MOE 951 147746 60765 60776 TAAAAACAACAA1-10-1 MOE 1073 384545 60822 60833 CAAGTAGGATGT 1-10-1 MOE 951 14768960967 60978 CAGAGAAGGTCT 1-10-1 MOE 987 147689 61008 61019 CAGAGAAGGTCT1-10-1 MOE 987 147689 61049 61060 CAGAGAAGGTCT 1-10-1 MOE 987 39810561121 61134 TGCACAGGCAGGTT 2-10-2 MOE 1066 147689 61154 61165CAGAGAAGGTCT 1-10-1 MOE 987 147689 61195 61206 CAGAGAAGGTCT 1-10-1 MOE987 398105 61267 61280 TGCACAGGCAGGTT 2-10-2 MOE 1066 147692 61365 61376CTCACCTTCATG 1-10-1 MOE 1113 147692 61511 61522 CTCACCTTCATG 1-10-1 MOE1113 147680 61619 61630 GTATGCACTGCT 1-10-1 MOE 988 147078 61755 61766CCTTCCACTGAT 1-10-1 MOE 1044 147079 61756 61767 TCCTTCCACTGA 1-10-1 MOE1001 147080 61757 61768 CTCCTTCCACTG 1-10-1 MOE 1021 147078 61901 61912CCTTCCACTGAT 1-10-1 MOE 1044 147079 61902 61913 TCCTTCCACTGA 1-10-1 MOE1001 147080 61903 61914 CTCCTTCCACTG 1-10-1 MOE 1021 147088 62361 62372CCCTCTACACCA 1-10-1 MOE 1050 401384 62573 62586 TGAACACATCACTA 2-10-2MOE 933 147688 62697 62708 TCCCAAACAAAT 1-10-1 MOE 990 147746 6310263113 TAAAAACAACAA 1-10-1 MOE 1073 147721 63225 63236 AATGCAGGATCT1-10-1 MOE 1118 147742 63226 63237 AACTTCAGTGTC 1-10-1 MOE 1041 14774663248 63259 TAAAAACAACAA 1-10-1 MOE 1073 147682 63337 63348 CGGGTACTATGG1-10-1 MOE 992 147721 63371 63382 AATGCAGGATCT 1-10-1 MOE 1118 14774263372 63383 AACTTCAGTGTC 1-10-1 MOE 1041 147688 63401 63412 TCCCAAACAAAT1-10-1 MOE 990 147097 63449 63460 GTTGTTGTTCCC 1-10-1 MOE 1111 14709863450 63461 AGTTGTTGTTCC 1-10-1 MOE 1112 401409 63458 63471ATTCTTAACACAGA 2-10-2 MOE 991 147084 63531 63542 CTACACCAGGTC 1-10-1 MOE993 147688 63547 63558 TCCCAAACAAAT 1-10-1 MOE 990 147097 63595 63606GTTGTTGTTCCC 1-10-1 MOE 1111 147098 63596 63607 AGTTGTTGTTCC 1-10-1 MOE1112 147721 64086 64097 AATGCAGGATCT 1-10-1 MOE 1118 147721 64232 64243AATGCAGGATCT 1-10-1 MOE 1118 147692 64233 64244 CTCACCTTCATG 1-10-1 MOE1113 147692 64379 64390 CTCACCTTCATG 1-10-1 MOE 1113 147729 64633 64644GTAAGAGGCAGG 1-10-1 MOE 920 401403 64746 64759 TTTCCTAGGAGGTG 2-10-2 MOE967 147729 64779 64790 GTAAGAGGCAGG 1-10-1 MOE 920 147746 65151 65162TAAAAACAACAA 1-10-1 MOE 1073 147746 65297 65308 TAAAAACAACAA 1-10-1 MOE1073 147689 65302 65313 CAGAGAAGGTCT 1-10-1 MOE 987 147689 65448 65459CAGAGAAGGTCT 1-10-1 MOE 987 147717 65862 65873 ATCTTCAGAGAT 1-10-1 MOE996 147717 65895 65906 ATCTTCAGAGAT 1-10-1 MOE 996 147729 66000 66011GTAAGAGGCAGG 1-10-1 MOE 920 147717 66008 66019 ATCTTCAGAGAT 1-10-1 MOE996 147717 66041 66052 ATCTTCAGAGAT 1-10-1 MOE 996 147708 66046 66057TTGATATAGTCA 1-10-1 MOE 997 147718 66055 66066 TAATATGACTTG 1-10-1 MOE998 147729 66146 66157 GTAAGAGGCAGG 1-10-1 MOE 920 147089 66236 66247TCCCTCTACACC 1-10-1 MOE 956 368363 66281 66294 CTTAGAAGGCAGCA 2-10-2 MOE1114 147727 66293 66304 CAGTGGACCACA 1-10-1 MOE 1128 147093 66319 66330TTGTTCCCTCTA 1-10-1 MOE 929 147094 66320 66331 GTTGTTCCCTCT 1-10-1 MOE1115 147089 66382 66393 TCCCTCTACACC 1-10-1 MOE 956 368363 66427 66440CTTAGAAGGCAGCA 2-10-2 MOE 1114 147727 66439 66450 CAGTGGACCACA 1-10-1MOE 1128 147719 66441 66452 CCAACTCCAACT 1-10-1 MOE 1116 147093 6646566476 TTGTTCCCTCTA 1-10-1 MOE 929 147094 66466 66477 GTTGTTCCCTCT 1-10-1MOE 1115 147075 66561 66572 TCCACTGATCCT 1-10-1 MOE 1026 368357 6656266575 CCTTCCACTGATCC 2-10-2 MOE 1046 147076 66562 66573 TTCCACTGATCC1-10-1 MOE 1029 368377 66562 66577 CTCCTTCCACTGA 3-10-3 MOE 1030 TCC147077 66563 66574 CTTCCACTGATC 1-10-1 MOE 1047 368358 66563 66576TCCTTCCACTGATC 2-10-2 MOE 1031 147078 66564 66575 CCTTCCACTGAT 1-10-1MOE 1044 147079 66565 66576 TCCTTCCACTGA 1-10-1 MOE 1001 147080 6656666577 CTCCTTCCACTG 1-10-1 MOE 1021 147081 66567 66578 GCTCCTTCCACT1-10-1 MOE 1006 147719 66587 66598 CCAACTCCAACT 1-10-1 MOE 1116 14707566707 66718 TCCACTGATCCT 1-10-1 MOE 1026 368377 66708 66723CTCCTTCCACTGA 3-10-3 MOE 1030 TCC 147076 66708 66719 TTCCACTGATCC 1-10-1MOE 1029 368357 66708 66721 CCTTCCACTGATCC 2-10-2 MOE 1046 147077 6670966720 CTTCCACTGATC 1-10-1 MOE 1047 147078 66710 66721 CCTTCCACTGAT1-10-1 MOE 1044 147079 66711 66722 TCCTTCCACTGA 1-10-1 MOE 1001 14708066712 66723 CTCCTTCCACTG 1-10-1 MOE 1021 147081 66713 66724 GCTCCTTCCACT1-10-1 MOE 1006 147089 66842 66853 TCCCTCTACACC 1-10-1 MOE 956 14708966988 66999 TCCCTCTACACC 1-10-1 MOE 956 147075 66999 67010 TCCACTGATCCT1-10-1 MOE 1026 147075 67145 67156 TCCACTGATCCT 1-10-1 MOE 1026 14770567213 67224 CGGTTTTTGTTC 1-10-1 MOE 1002 401413 67301 67314TGCAGCCATGTACT 2-10-2 MOE 1022 147737 67309 67320 ACAGCCAGGTAG 1-10-1MOE 1067 147080 67430 67441 CTCCTTCCACTG 1-10-1 MOE 1021 147737 6745567466 ACAGCCAGGTAG 1-10-1 MOE 1067 147080 67576 67587 CTCCTTCCACTG1-10-1 MOE 1021 147082 67578 67589 AGCTCCTTCCAC 1-10-1 MOE 1036 14709067582 67593 TTCCCTCTACAC 1-10-1 MOE 955 147091 67583 67594 GTTCCCTCTACA1-10-1 MOE 1004 147742 67591 67602 AACTTCAGTGTC 1-10-1 MOE 1041 14709067728 67739 TTCCCTCTACAC 1-10-1 MOE 955 147698 68036 68047 CCCGCCACCACC1-10-1 MOE 928 147698 68182 68193 CCCGCCACCACC 1-10-1 MOE 928 14768168267 68278 ATGTCATTAAAC 1-10-1 MOE 965 147721 68386 68397 AATGCAGGATCT1-10-1 MOE 1118 147681 68413 68424 ATGTCATTAAAC 1-10-1 MOE 965 14771268527 68538 ACACCATCTCCC 1-10-1 MOE 1005 147721 68532 68543 AATGCAGGATCT1-10-1 MOE 1118 147711 68760 68771 AAGGGCCCTGGG 1-10-1 MOE 1040 14771168906 68917 AAGGGCCCTGGG 1-10-1 MOE 1040 147696 69045 69056 TGGATGATTGGC1-10-1 MOE 906 147696 69191 69202 TGGATGATTGGC 1-10-1 MOE 906 14772369194 69205 GACTCCAAAGTC 1-10-1 MOE 892 147723 69210 69221 GACTCCAAAGTC1-10-1 MOE 892 389965 69271 69282 CTGCAACATGAT 1-10-1 MOE 1018 38976469271 69282 CTGCAACATGAT 1-9-2 MOE 1018 147723 69340 69351 GACTCCAAAGTC1-10-1 MOE 892 147723 69356 69367 GACTCCAAAGTC 1-10-1 MOE 892 39810169357 69370 TTTGATAAAGCCCT 2-10-2 MOE 1064 389965 69417 69428CTGCAACATGAT 1-10-1 MOE 1018 389764 69417 69428 CTGCAACATGAT 1-9-2 MOE1018 398101 69503 69516 TTTGATAAAGCCCT 2-10-2 MOE 1064 368353 6951969532 CACTGATCCTGCAC 2-10-2 MOE 1007 147074 69522 69533 CCACTGATCCTG1-10-1 MOE 845 147081 69631 69642 GCTCCTTCCACT 1-10-1 MOE 1006 36835369665 69678 CACTGATCCTGCAC 2-10-2 MOE 1007 147720 69729 69740GATCTCTCGAGT 1-10-1 MOE 1117 147721 69736 69747 AATGCAGGATCT 1-10-1 MOE1118 398167 69757 69768 CAGGCCATGTGG 1-10-1 MOE 1059 147722 69762 69773AAAGTCAGGCCA 1-10-1 MOE 1130 147723 69768 69779 GACTCCAAAGTC 1-10-1 MOE892 147080 69776 69787 CTCCTTCCACTG 1-10-1 MOE 1021 147081 69777 69788GCTCCTTCCACT 1-10-1 MOE 1006 398093 69811 69824 TCGGACTTTGAAAA 2-10-2MOE 1009 398168 69813 69824 TCGGACTTTGAA 1-10-1 MOE 1008 147725 6981469825 CTCGGACTTTGA 1-10-1 MOE 1119 147726 69819 69830 TGACTCTCGGAC1-10-1 MOE 1120 147727 69860 69871 CAGTGGACCACA 1-10-1 MOE 1128 14772069875 69886 GATCTCTCGAGT 1-10-1 MOE 1117 147721 69882 69893 AATGCAGGATCT1-10-1 MOE 1118 147728 69899 69910 GCCAGACAGAAG 1-10-1 MOE 1013 39809469901 69914 ATCAGCCAGACAGA 2-10-2 MOE 1010 398167 69903 69914CAGGCCATGTGG 1-10-1 MOE 1059 398092 69904 69917 AGTCAGGCCATGTG 2-10-2MOE 1060 147722 69908 69919 AAAGTCAGGCCA 1-10-1 MOE 1130 147723 6991469925 GACTCCAAAGTC 1-10-1 MOE 892 147729 69916 69927 GTAAGAGGCAGG 1-10-1MOE 920 398095 69919 69932 CATCAGCAAGAGGC 2-10-2 MOE 1011 398093 6995769970 TCGGACTTTGAAAA 2-10-2 MOE 1009 398168 69959 69970 TCGGACTTTGAA1-10-1 MOE 1008 147725 69960 69971 CTCGGACTTTGA 1-10-1 MOE 1119 14772669965 69976 TGACTCTCGGAC 1-10-1 MOE 1120 147704 69991 70002 TTGTTCTTAGGA1-10-1 MOE 1012 147727 70006 70017 CAGTGGACCACA 1-10-1 MOE 1128 14772870045 70056 GCCAGACAGAAG 1-10-1 MOE 1013 398094 70047 70060ATCAGCCAGACAGA 2-10-2 MOE 1010 398169 70048 70059 TCAGCCAGACAG 1-10-1MOE 909 147729 70062 70073 GTAAGAGGCAGG 1-10-1 MOE 920 398095 7006570078 CATCAGCAAGAGGC 2-10-2 MOE 1011 147704 70137 70148 TTGTTCTTAGGA1-10-1 MOE 1012 147697 70161 70172 CCCCAGCAGCGG 1-10-1 MOE 1000 14769770307 70318 CCCCAGCAGCGG 1-10-1 MOE 1000 147728 70450 70461 GCCAGACAGAAG1-10-1 MOE 1013 398164 70464 70475 TTGTCGATCTGC 1-10-1 MOE 1014 14773070465 70476 CTTGTCCATCAG 1-10-1 MOE 1121 147731 70471 70482 TTTCCTCTTGTC1-10-1 MOE 934 147732 70476 70487 GGGTCTTTCCTC 1-10-1 MOE 1122 14773370497 70508 TTCTTGATGTCC 1-10-1 MOE 891 398096 70562 70575GGAGAAGCGCAGCT 2-10-2 MOE 1015 147735 70564 70575 GGAGAAGCGCAG 1-10-1MOE 1016 147736 70569 70580 AGGTAGGAGAAG 1-10-1 MOE 963 147737 7057570586 ACAGCCAGGTAG 1-10-1 MOE 1067 147728 70596 70607 GCCAGACAGAAG1-10-1 MOE 1013 398164 70610 70621 TTGTCGATCTGC 1-10-1 MOE 1014 14773070611 70622 CTTGTCCATCAG 1-10-1 MOE 1121 368349 70616 70629CTGCACTGACGAGT 2-10-2 MOE 1017 147731 70617 70628 TTTCCTCTTGTC 1-10-1MOE 934 147732 70622 70633 GGGTCTTTCCTC 1-10-1 MOE 1122 147733 7064370654 TTCTTGATGTCC 1-10-1 MOE 891 398096 70708 70721 GGAGAAGCGCAGCT2-10-2 MOE 1015 147735 70710 70721 GGAGAAGCGCAG 1-10-1 MOE 1016 14773670715 70726 AGGTAGGAGAAG 1-10-1 MOE 963 147737 70721 70732 ACAGCCAGGTAG1-10-1 MOE 1067 389764 70784 70795 CTGCAACATGAT 1-9-2 MOE 1018 38996570784 70795 CTGCAACATGAT 1-10-1 MOE 1018 389965 70930 70941 CTGCAACATGAT1-10-1 MOE 1018 389764 70930 70941 CTGCAACATGAT 1-9-2 MOE 1018 36838670995 71010 CACTGATCCTTAG 3-10-3 MOE 1123 AAG 368367 70997 71010CACTGATCCTTAGA 2-10-2 MOE 1124 368387 70997 71012 TCCACTGATCCTT 3-10-3MOE 1125 AGA 368354 70999 71012 TCCACTGATCCTGC 2-10-2 MOE 1024 36837470999 71014 CTTCCACTGATCC 3-10-3 MOE 1126 TGC 368368 70999 71012TCCACTGATCCTTA 2-10-2 MOE 1127 368388 70999 71014 CTTCCACTGATCC 3-10-3MOE 895 TTA 368355 71000 71013 TTCCACTGATCCTG 2-10-2 MOE 1025 14707471000 71011 CCACTGATCCTG 1-10-1 MOE 845 368375 71000 71015 CCTTCCACTGATC3-10-3 MOE 1020 CTG 147075 71001 71012 TCCACTGATCCT 1-10-1 MOE 1026368376 71001 71016 TCCTTCCACTGAT 3-10-3 MOE 1028 CCT 147076 71002 71013TTCCACTGATCC 1-10-1 MOE 1029 368357 71002 71015 CCTTCCACTGATCC 2-10-2MOE 1046 368377 71002 71017 CTCCTTCCACTGA 3-10-3 MOE 1030 TCC 14707771003 71014 CTTCCACTGATC 1-10-1 MOE 1047 368378 71003 71018GCTCCTTCCACTG 3-10-3 MOE 1032 ATC 147078 71004 71015 CCTTCCACTGAT 1-10-1MOE 1044 368359 71005 71018 GCTCCTTCCACTGA 2-10-2 MOE 1033 368379 7100571020 AAGCTCCTTCCAC 3-10-3 MOE 1034 TGA 147079 71005 71016 TCCTTCCACTGA1-10-1 MOE 1001 147080 71006 71017 CTCCTTCCACTG 1-10-1 MOE 1021 36836071007 71020 AAGCTCCTTCCACT 2-10-2 MOE 1035 368380 71007 71022GAAAGCTCCTTCC 3-10-3 MOE 896 ACT 147081 71007 71018 GCTCCTTCCACT 1-10-1MOE 1006 147082 71008 71019 AGCTCCTTCCAC 1-10-1 MOE 1036 368361 7100971022 GAAAGCTCCTTCCA 2-10-2 MOE 962 368381 71009 71024 GGGAAAGCTCCTT3-10-3 MOE 1037 CCA 147738 71067 71078 TGGGTGGCCGGG 1-10-1 MOE 1069147739 71071 71082 CGTTTGGGTGGC 1-10-1 MOE 1023 147740 71088 71099TGTGAGGCTCCA 1-10-1 MOE 1062 147741 71129 71140 CACCCACTGGTG 1-10-1 MOE1055 368366 71141 71154 CTGATCCTTAGAAG 2-10-2 MOE 1019 368386 7114171156 CACTGATCCTTAG 3-10-3 MOE 1123 AAG 368367 71143 71156CACTGATCCTTAGA 2-10-2 MOE 1124 368387 71143 71158 TCCACTGATCCTT 3-10-3MOE 1125 AGA 368374 71145 71160 CTTCCACTGATCC 3-10-3 MOE 1126 TGC 36835471145 71158 TCCACTGATCCTGC 2-10-2 MOE 1024 368368 71145 71158TCCACTGATCCTTA 2-10-2 MOE 1127 368388 71145 71160 CTTCCACTGATCC 3-10-3MOE 895 TTA 368355 71146 71159 TTCCACTGATCCTG 2-10-2 MOE 1025 36837571146 71161 CCTTCCACTGATC 3-10-3 MOE 1020 CTG 147075 71147 71158TCCACTGATCCT 1-10-1 MOE 1026 368356 71147 71160 CTTCCACTGATCCT 2-10-2MOE 1027 368376 71147 71162 TCCTTCCACTGAT 3-10-3 MOE 1028 CCT 14707671148 71159 TTCCACTGATCC 1-10-1 MOE 1029 368357 71148 71161CCTTCCACTGATCC 2-10-2 MOE 1046 368377 71148 71163 CTCCTTCCACTGA 3-10-3MOE 1030 TCC 147077 71149 71160 CTTCCACTGATC 1-10-1 MOE 1047 36835871149 71162 TCCTTCCACTGATC 2-10-2 MOE 1031 368378 71149 71164GCTCCTTCCACTG 3-10-3 MOE 1032 ATC 147078 71150 71161 CCTTCCACTGAT 1-10-1MOE 1044 368359 71151 71164 GCTCCTTCCACTGA 2-10-2 MOE 1033 147079 7115171162 TCCTTCCACTGA 1-10-1 MOE 1001 368379 71151 71166 AAGCTCCTTCCAC3-10-3 MOE 1034 TGA 147080 71152 71163 CTCCTTCCACTG 1-10-1 MOE 1021368380 71153 71168 GAAAGCTCCTTCC 3-10-3 MOE 896 ACT 147081 71153 71164GCTCCTTCCACT 1-10-1 MOE 1006 368360 71153 71166 AAGCTCCTTCCACT 2-10-2MOE 1035 147082 71154 71165 AGCTCCTTCCAC 1-10-1 MOE 1036 368381 7115571170 GGGAAAGCTCCTT 3-10-3 MOE 1037 CCA 368361 71155 71168GAAAGCTCCTTCCA 2-10-2 MOE 962 398097 71158 71171 GGCAGTCTTTATCC 2-10-2MOE 897 147738 71213 71224 TGGGTGGCCGGG 1-10-1 MOE 1069 147739 7121771228 CGTTTGGGTGGC 1-10-1 MOE 1023 147740 71234 71245 TGTGAGGCTCCA1-10-1 MOE 1062 147741 71275 71286 CACCCACTGGTG 1-10-1 MOE 1055 39809771304 71317 GGCAGTCTTTATCC 2-10-2 MOE 897 147727 71702 71713CAGTGGACCACA 1-10-1 MOE 1128 147727 71848 71859 CAGTGGACCACA 1-10-1 MOE1128 390030 71986 71997 TTTATAAAACTG 1-10-1 MOE 1074 147102 72015 72026TGCGAGTTGTTG 1-10-1 MOE 1129 390030 72132 72143 TTTATAAAACTG 1-10-1 MOE1074 147102 72161 72172 TGCGAGTTGTTG 1-10-1 MOE 1129 147722 72199 72210AAAGTCAGGCCA 1-10-1 MOE 1130 147696 72232 72243 TGGATGATTGGC 1-10-1 MOE906 147741 72254 72265 CACCCACTGGTG 1-10-1 MOE 1055 147722 72345 72356AAAGTCAGGCCA 1-10-1 MOE 1130 147696 72378 72389 TGGATGATTGGC 1-10-1 MOE906 147741 72400 72411 CACCCACTGGTG 1-10-1 MOE 1055 147711 72446 72457AAGGGCCCTGGG 1-10-1 MOE 1040 398098 72574 72587 TAACTTCAGTGTCT 2-10-2MOE 1131 147742 72575 72586 AACTTCAGTGTC 1-10-1 MOE 1041 147698 7259572606 CCCGCCACCACC 1-10-1 MOE 928 147743 72690 72701 AGGGCTTCCAGT 1-10-1MOE 1042 398099 72690 72703 GAAGGGCTTCCAGT 2-10-2 MOE 1132 147744 7269472705 AGGAAGGGCTTC 1-10-1 MOE 1043 398100 72697 72710 TGACCAGGAAGGGC2-10-2 MOE 1133 147745 72700 72711 TTGACCAGGAAG 1-10-1 MOE 1058 39809872720 72733 TAACTTCAGTGTCT 2-10-2 MOE 1131 147742 72721 72732AACTTCAGTGTC 1-10-1 MOE 1041 147698 72741 72752 CCCGCCACCACC 1-10-1 MOE928 398157 72757 72770 GGAAACATACCCTG 2-10-2 MOE 1045 147743 72836 72847AGGGCTTCCAGT 1-10-1 MOE 1042 398099 72836 72849 GAAGGGCTTCCAGT 2-10-2MOE 1132 147744 72840 72851 AGGAAGGGCTTC 1-10-1 MOE 1043 398100 7284372856 TGACCAGGAAGGGC 2-10-2 MOE 1133 147745 72846 72857 TTGACCAGGAAG1-10-1 MOE 1058 147076 72898 72909 TTCCACTGATCC 1-10-1 MOE 1029 36835772898 72911 CCTTCCACTGATCC 2-10-2 MOE 1046 147077 72899 72910CTTCCACTGATC 1-10-1 MOE 1047 147078 72900 72911 CCTTCCACTGAT 1-10-1 MOE1044 398157 72903 72916 GGAAACATACCCTG 2-10-2 MOE 1045 398158 7298372996 AGGCCCTGAGATTA 2-10-2 MOE 1134 398159 72988 73001 GGTTAAGGCCCTGA2-10-2 MOE 1135 398160 72993 73006 GAATAGGTTAAGGC 2-10-2 MOE 1048 14707673044 73055 TTCCACTGATCC 1-10-1 MOE 1029 368357 73044 73057CCTTCCACTGATCC 2-10-2 MOE 1046 147077 73045 73056 CTTCCACTGATC 1-10-1MOE 1047 147078 73046 73057 CCTTCCACTGAT 1-10-1 MOE 1044 147746 7305273063 TAAAAACAACAA 1-10-1 MOE 1073 398161 73092 73105 AACAATGTGTTGTA2-10-2 MOE 1049 147746 73101 73112 TAAAAACAACAA 1-10-1 MOE 1073 39815873129 73142 AGGCCCTGAGATTA 2-10-2 MOE 1134 398159 73134 73147GGTTAAGGCCCTGA 2-10-2 MOE 1135 398160 73139 73152 GAATAGGTTAAGGC 2-10-2MOE 1048 147746 73198 73209 TAAAAACAACAA 1-10-1 MOE 1073 398161 7323873251 AACAATGTGTTGTA 2-10-2 MOE 1049 147746 73247 73258 TAAAAACAACAA1-10-1 MOE 1073 147088 73273 73284 CCCTCTACACCA 1-10-1 MOE 1050 39810573401 73414 TGCACAGGCAGGTT 2-10-2 MOE 1066 398105 73547 73560TGCACAGGCAGGTT 2-10-2 MOE 1066 147741 73559 73570 CACCCACTGGTG 1-10-1MOE 1055 147741 73705 73716 CACCCACTGGTG 1-10-1 MOE 1055 398162 7396873981 ACCAAACAGTTCAG 2-10-2 MOE 1057 147745 73991 74002 TTGACCAGGAAG1-10-1 MOE 1058 398167 74008 74019 CAGGCCATGTGG 1-10-1 MOE 1059 39809274009 74022 AGTCAGGCCATGTG 2-10-2 MOE 1060 398162 74114 74127ACCAAACAGTTCAG 2-10-2 MOE 1057 147745 74137 74148 TTGACCAGGAAG 1-10-1MOE 1058 398167 74154 74165 CAGGCCATGTGG 1-10-1 MOE 1059 147089 7428074291 TCCCTCTACACC 1-10-1 MOE 956 147090 74281 74292 TTCCCTCTACAC 1-10-1MOE 955 389949 74310 74321 GCGCGAGCCCGA 1-10-1 MOE 1061 147740 7433974350 TGTGAGGCTCCA 1-10-1 MOE 1062 389950 74381 74392 CCCTGAAGGTTC1-10-1 MOE 1063 147089 74426 74437 TCCCTCTACACC 1-10-1 MOE 956 14709074427 74438 TTCCCTCTACAC 1-10-1 MOE 955 389949 74456 74467 GCGCGAGCCCGA1-10-1 MOE 1061 147685 74490 74501 GGCTGACATTCA 1-10-1 MOE 975 39810174510 74523 TTTGATAAAGCCCT 2-10-2 MOE 1064 398102 74536 74549CTACCTGAGGATTT 2-10-2 MOE 899 398103 74543 74556 CCCAGTACTACCTG 2-10-2MOE 900 147685 74636 74647 GGCTGACATTCA 1-10-1 MOE 975 398102 7468274695 CTACCTGAGGATTT 2-10-2 MOE 899 398103 74689 74702 CCCAGTACTACCTG2-10-2 MOE 900 147736 74737 74748 AGGTAGGAGAAG 1-10-1 MOE 963 39810474805 74818 CAAGAAGACCTTAC 2-10-2 MOE 1065 147736 74883 74894AGGTAGGAGAAG 1-10-1 MOE 963 147737 74893 74904 ACAGCCAGGTAG 1-10-1 MOE1067 398105 74894 74907 TGCACAGGCAGGTT 2-10-2 MOE 1066 147737 7491974930 ACAGCCAGGTAG 1-10-1 MOE 1067 398095 74940 74953 CATCAGCAAGAGGC2-10-2 MOE 1011 398104 74951 74964 CAAGAAGACCTTAC 2-10-2 MOE 1065 39810674974 74987 TGGAAAACTGCACC 2-10-2 MOE 1068 398107 74980 74993TATTCCTGGAAAAC 2-10-2 MOE 902 147745 75030 75041 TTGACCAGGAAG 1-10-1 MOE1058 147737 75039 75050 ACAGCCAGGTAG 1-10-1 MOE 1067 398105 75040 75053TGCACAGGCAGGTT 2-10-2 MOE 1066 147737 75065 75076 ACAGCCAGGTAG 1-10-1MOE 1067 398108 75077 75090 GGAATGTCTGAGTT 2-10-2 MOE 1136 398095 7508675099 CATCAGCAAGAGGC 2-10-2 MOE 1011 147691 75108 75119 GAGGTGGGAAAA1-10-1 MOE 966 398106 75120 75133 TGGAAAACTGCACC 2-10-2 MOE 1068 39810775126 75139 TATTCCTGGAAAAC 2-10-2 MOE 902 147738 75155 75166TGGGTGGCCGGG 1-10-1 MOE 1069 147745 75176 75187 TTGACCAGGAAG 1-10-1 MOE1058 398108 75223 75236 GGAATGTCTGAGTT 2-10-2 MOE 1136 398109 7524775260 CAAGAAGTGTGGTT 2-10-2 MOE 903 147691 75254 75265 GAGGTGGGAAAA1-10-1 MOE 966 147738 75301 75312 TGGGTGGCCGGG 1-10-1 MOE 1069 39811075385 75398 GTTCCCTTTGCAGG 2-10-2 MOE 952 147091 75387 75398GTTCCCTCTACA 1-10-1 MOE 1004 398109 75393 75406 CAAGAAGTGTGGTT 2-10-2MOE 903 398111 75470 75483 GTGAAAATGCTGGC 2-10-2 MOE 904 401385 7549475507 CCCAGTGGGTTTGA 2-10-2 MOE 890 398166 75499 75510 GGGCTTCTTCCA1-10-1 MOE 1070 147091 75525 75536 GTTCCCTCTACA 1-10-1 MOE 1004 14709275526 75537 TGTTCCCTCTAC 1-10-1 MOE 901 398110 75531 75544GTTCCCTTTGCAGG 2-10-2 MOE 952 147091 75533 75544 GTTCCCTCTACA 1-10-1 MOE1004 147706 75540 75551 GCTGACATCTCG 1-10-1 MOE 1071 398112 75584 75597CAGCCTGGCACCTA 2-10-2 MOE 1072 398111 75616 75629 GTGAAAATGCTGGC 2-10-2MOE 904 147746 75617 75628 TAAAAACAACAA 1-10-1 MOE 1073 398166 7564575656 GGGCTTCTTCCA 1-10-1 MOE 1070 147091 75671 75682 GTTCCCTCTACA1-10-1 MOE 1004 147092 75672 75683 TGTTCCCTCTAC 1-10-1 MOE 901 39811375693 75706 AGGAGGTTAAACCA 2-10-2 MOE 905 398112 75730 75743CAGCCTGGCACCTA 2-10-2 MOE 1072 147746 75763 75774 TAAAAACAACAA 1-10-1MOE 1073 398114 75770 75783 AGGCATATAGCAGA 2-10-2 MOE 1075 398115 7578675799 AGTAAATATTGGCT 2-10-2 MOE 1076 398116 75799 75812 TAATGACCTGATGA2-10-2 MOE 1137 398113 75839 75852 AGGAGGTTAAACCA 2-10-2 MOE 905 39003075839 75850 TTTATAAAACTG 1-10-1 MOE 1074 398115 75932 75945AGTAAATATTGGCT 2-10-2 MOE 1076 398116 75945 75958 TAATGACCTGATGA 2-10-2MOE 1137 398106 75982 75995 TGGAAAACTGCACC 2-10-2 MOE 1068 390030 7598575996 TTTATAAAACTG 1-10-1 MOE 1074 398106 76127 76140 TGGAAAACTGCACC2-10-2 MOE 1068 147690 76196 76207 TGAAGTTAATTC 1-10-1 MOE 1138 14769076341 76352 TGAAGTTAATTC 1-10-1 MOE 1138 147724 76740 76751 GAAATTGAGGAA1-10-1 MOE 1139 147089 76873 76884 TCCCTCTACACC 1-10-1 MOE 956 14767976881 76892 CAAAAGGATCCC 1-10-1 MOE 907 147724 76885 76896 GAAATTGAGGAA1-10-1 MOE 1139 147089 77018 77029 TCCCTCTACACC 1-10-1 MOE 956 14767977026 77037 CAAAAGGATCCC 1-10-1 MOE 907 147693 77240 77251 GTGCGCTCCCAT1-10-1 MOE 1078 147697 77759 77770 CCCCAGCAGCGG 1-10-1 MOE 1000

In certain embodiments, a target region is nucleotides 177-190 of SEQ IDNO: 11. In certain embodiments, a short antisense compound is targetedto nucleotides 177-190 of SEQ ID NO: 11. In certain such embodiments, ashort antisense compound targeted to nucleotides 177-190 comprises anucleotide sequence selected from SEQ ID NO 886, 859, or 853. In certainsuch embodiments, a short antisense compound targeted to nucleotides177-190 of SEQ ID NO: 11 is selected from Isis No 147022, 147023, or147024.

In certain embodiments, a target region is nucleotides 195-228 of SEQ IDNO: 11. In certain embodiments, a short antisense compound is targetedto nucleotides 195-228 of SEQ ID NO: 11. In certain such embodiments, ashort antisense compound targeted to nucleotides 195-228 comprises anucleotide sequence selected from SEQ ID NO 877, 868, 882, 886, 859,853, 865, 835, 843, 846, 842, 848, 874, 849, 863, 855, 850, 864, or 834.In certain such embodiments, a short antisense compound targeted tonucleotides 195-228 of SEQ ID NO: 11 is selected from Isis No 147019,147020, 147021, 147022, 147023, 147024, 147025, 147026, 147027, 147028,147073, 147029, 147030, 147036, 147037, 147038, 147039, 147040, or147041.

In certain embodiments, a target region is nucleotides 323-353 of SEQ IDNO: 11. In certain embodiments, a short antisense compound is targetedto nucleotides 323-353 of SEQ ID NO: 11. In certain such embodiments, ashort antisense compound targeted to nucleotides 323-353 comprises anucleotide sequence selected from SEQ ID NO 866, 881, 869, 883, 858,833, 875, 837, 829, 871, 884, 887, 839, 830, 840, 861, or 879. Incertain such embodiments, a short antisense compound targeted tonucleotides 323-353 of SEQ ID NO: 11 is selected from Isis No 147042,147043, 147044, 147045, 147046, 147047, 147051, 147052, 147053, 147054,147055, 147056, 147057, 147058, 147059, 147060, or 147061.

In certain embodiments, a target region is nucleotides 322-353 of SEQ IDNO: 11. In certain embodiments, a short antisense compound is targetedto nucleotides 322-353 of SEQ ID NO: 11. In certain such embodiments, ashort antisense compound targeted to nucleotides 322-353 comprises anucleotide sequence selected from SEQ ID NO 842, 866, 881, 869, 883,858, 833, 875, 837, 829, 871, 884, 887, 839, 830, 840, 861, or 879. Incertain such embodiments, a short antisense compound targeted tonucleotides 322-353 of SEQ ID NO: 11 is selected from Isis No 147073,147042, 147043, 147044, 147045, 147046, 147047, 147051, 147052, 147053,147054, 147055, 147056, 147057, 147058, 147059, 147060, or 147061.

In certain embodiments, a target region is nucleotides 679-799 of SEQ IDNO: 11. In certain embodiments, a short antisense compound is targetedto nucleotides 679-799 of SEQ ID NO: 11. In certain such embodiments, ashort antisense compound targeted to nucleotides 679-799 comprises anucleotide sequence selected from SEQ ID NO 883, 858, 883, or 858. Incertain such embodiments, a short antisense compound targeted tonucleotides 679-799 of SEQ ID NO: 11 is selected from Isis No 147045,147046, 147045, or 147046.

In certain embodiments, a target region is nucleotides 679-827 of SEQ IDNO: 11. In certain embodiments, a short antisense compound is targetedto nucleotides 679-827 of SEQ ID NO: 11. In certain such embodiments, ashort antisense compound targeted to nucleotides 679-827 comprises anucleotide sequence selected from SEQ ID NO 883, 858, 883, 858, or 851.In certain such embodiments, a short antisense compound targeted tonucleotides 679-827 of SEQ ID NO: 11 is selected from Isis No 147045,147046, 147045, 147046, or 147066.

In certain embodiments, a target region is nucleotides 1024-1046 of SEQID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 1024-1046 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to nucleotides1024-1046 comprises a nucleotide sequence selected from SEQ ID NO 841,862, 880, 857, 851, 876, 838, 860, 878, 856, 832, or 842. In certainsuch embodiments, a short antisense compound targeted to nucleotides1024-1046 of SEQ ID NO: 11 is selected from Isis No 147062, 147063,147064, 147065, 147066, 147067, 147068, 147069, 147070, 147071, 147072,or 147073.

In certain embodiments, a target region is nucleotides 992-1046 of SEQID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 992-1046 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to nucleotides 992-1046comprises a nucleotide sequence selected from SEQ ID NO 831, 841, 862,880, 857, 851, 876, 838, 860, 878, 856, 832, or 842. In certain suchembodiments, a short antisense compound targeted to nucleotides 992-1046of SEQ ID NO: 11 is selected from Isis No 404131, 147062, 147063,147064, 147065, 147066, 147067, 147068, 147069, 147070, 147071, 147072,or 147073.

In certain embodiments, a target region is nucleotides 1868-1881 of SEQID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 1868-1881 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to nucleotides1868-1881 comprises a nucleotide sequence selected from SEQ ID NO 886,859, or 853. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1868-1881 of SEQ ID NO: 11 is selected from IsisNo 147022, 147023, or 147024.

In certain embodiments, a target region is nucleotides 1886-1919 of SEQID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 1886-1919 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to nucleotides1886-1919 comprises a nucleotide sequence selected from SEQ ID NO 877,868, 882, 886, 859, 865, 843, 846, 874, 863, 855, 864, or 834. Incertain such embodiments, a short antisense compound targeted tonucleotides 1886-1919 of SEQ ID NO: 11 is selected from Isis No 147019,147020, 147021, 147022, 147023, 147025, 147027, 147028, 147030, 147037,147038, 147040, or 147041.

In certain embodiments, a target region is nucleotides 1869-1919 of SEQID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 1869-1919 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to nucleotides1869-1919 comprises a nucleotide sequence selected from SEQ ID NO 859,853, 877, 868, 882, 886, 859, 865, 843, 846, 874, 863, 855, 864, or 834.In certain such embodiments, a short antisense compound targeted tonucleotides 1869-1919 of SEQ ID NO: 11 is selected from Isis No 147023,147024, 147019, 147020, 147021, 147022, 147023, 147025, 147027, 147028,147030, 147037, 147038, 147040, or 147041.

In certain embodiments, a target region is nucleotides 1976-1989 of SEQID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 1976-1989 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to nucleotides1976-1989 comprises a nucleotide sequence selected from SEQ ID NO 886,859, or 853. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1976-1989 of SEQ ID NO: 11 is selected from IsisNo 147022, 147023, or 147024.

In certain embodiments, a target region is nucleotides 1995-2027 of SEQID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 1995-2027 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to nucleotides1995-2027 comprises a nucleotide sequence selected from SEQ ID NO 868,882, 886, 859, 853, 865, 835, 843, 846, 848, 874, 849, 863, 855, 850,864, or 834. In certain such embodiments, a short antisense compoundtargeted to nucleotides 1995-2027 of SEQ ID NO: 11 is selected from IsisNo 147020, 147021, 147022, 147023, 147024, 147025, 147026, 147027,147028, 147029, 147030, 147036, 147037, 147038, 147039, 147040, or147041.

In certain embodiments, a target region is nucleotides 2366-2382 of SEQID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 2366-2382 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to nucleotides2366-2382 comprises a nucleotide sequence selected from SEQ ID NO 867 or873. In certain such embodiments, a short antisense compound targeted tonucleotides 2366-2382 of SEQ ID NO: 11 is selected from Isis No 404199or 404134.

In certain embodiments, a target region is nucleotides 6220-6233 of SEQID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 6220-6233 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to nucleotides6220-6233 comprises a nucleotide sequence selected from SEQ ID NO 870,836, or 844. In certain such embodiments, a short antisense compoundtargeted to nucleotides 6220-6233 of SEQ ID NO: 11 is selected from IsisNo 147032, 147033, or 147034.

In certain embodiments, a target region is nucleotides 6288-6300 of SEQID NO: 1. In certain embodiments, a short antisense compound is targetedto nucleotides 6288-6300 of SEQ ID NO: 11. In certain such embodiments,a short antisense compound targeted to nucleotides 6288-6300 comprises anucleotide sequence selected from SEQ ID NO 869 or 883. In certain suchembodiments, a short antisense compound targeted to nucleotides6288-6300 of SEQ ID NO: 11 is selected from Isis No 147044 or 147045.

In certain embodiments, a target region is nucleotides 6329-6342 of SEQID NO: 1. In certain embodiments, a short antisense compound is targetedto nucleotides 6329-6342 of SEQ ID NO: 1. In certain such embodiments, ashort antisense compound targeted to nucleotides 6329-6342 comprises anucleotide sequence selected from SEQ ID NO 870, 836, or 844. In certainsuch embodiments, a short antisense compound targeted to nucleotides6329-6342 of SEQ ID NO: 11 is selected from Isis No 147032, 147033, or147034.

In certain embodiments, a target region is nucleotides 6397-6409 of SEQID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 6397-6409 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to nucleotides6397-6409 comprises a nucleotide sequence selected from SEQ ID NO 869 or883. In certain such embodiments, a short antisense compound targeted tonucleotides 6397-6409 of SEQ ID NO: 11 is selected from Isis No 147044or 147045.

In certain embodiments, a target region is nucleotides 7057-7178 of SEQID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 7057-7178 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to 7057-7178 comprisesa nucleotide sequence selected from SEQ ID NO 830, 840, 861, 830, or840. In certain such embodiments, a short antisense compound targeted tonucleotides 7057-7178 of SEQ ID NO: 11 is selected from Isis No 147058,147059, 147060, 147058, or 147059.

In certain embodiments, a target region is nucleotides 8630-8750 of SEQID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 8630-8750 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to 8630-8750 comprisesa nucleotide sequence selected from SEQ ID NO 843, 846, 843, or 846. Incertain such embodiments, a short antisense compound targeted tonucleotides 8630-8750 of SEQ ID NO: 11 is selected from Isis No 147027,147028, 147027, or 147028.

In certain embodiments, a target region is nucleotides 10957-11077 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 10957-11077 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to 10957-11077comprises a nucleotide sequence selected from SEQ ID NO 881, 869, 881,or 869. In certain such embodiments, a short antisense compound targetedto nucleotides 10957-11077 of SEQ ID NO: 11 is selected from Isis No147043, 147044, 147043, or 147044.

In certain embodiments, a target region is nucleotides 11605-11623 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 11605-11623 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to 11605-11623comprises a nucleotide sequence selected from SEQ ID NO 856, 878, or856. In certain such embodiments, a short antisense compound targeted tonucleotides 11605-11623 of SEQ ID NO: 11 is selected from Isis No147071, 147070, or 147071.

In certain embodiments, a target region is nucleotides 12805-12817 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 12805-12817 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to 12805-12817comprises a nucleotide sequence selected from SEQ ID NO 874 or 885. Incertain such embodiments, a short antisense compound targeted tonucleotides 12805-12817 of SEQ ID NO: 11 is selected from Isis No 147030or 147031.

In certain embodiments, a target region is nucleotides 12986-12998 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 12986-12998 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to 12986-12998comprises a nucleotide sequence selected from SEQ ID NO 874 or 885. Incertain such embodiments, a short antisense compound targeted tonucleotides 12986-12998 of SEQ ID NO: 11 is selected from Isis No 147030or 147031.

In certain embodiments, a target region is nucleotides 15560-15572 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 15560-15572 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to 15560-15572comprises a nucleotide sequence selected from SEQ ID NO 876 or 838. Incertain such embodiments, a short antisense compound targeted tonucleotides 15560-15572 of SEQ ID NO: 11 is selected from Isis No 147067or 147068.

In certain embodiments, a target region is nucleotides 17787-17941 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 17787-17941 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to 17787-17941comprises a nucleotide sequence selected from SEQ ID NO 874 or 880. Incertain such embodiments, a short antisense compound targeted tonucleotides 17787-17941 of SEQ ID NO: 11 is selected from Isis No 147030or 147064.

In certain embodiments, a target region is nucleotides 21190-21202 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 21190-21202 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to 21190-21202comprises a nucleotide sequence selected from SEQ ID NO 843 or 846. Incertain such embodiments, a short antisense compound targeted tonucleotides 21190-21202 of SEQ ID NO: 11 is selected from Isis No 147027or 147028.

In certain embodiments, a target region is nucleotides 21358-21370 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 21358-21370 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to 21358-21370comprises a nucleotide sequence selected from SEQ ID NO 843 or 846. Incertain such embodiments, a short antisense compound targeted tonucleotides 21358-21370 of SEQ ID NO: 11 is selected from Isis No 017027or 147028.

In certain embodiments, a target region is nucleotides 24318-24332 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 24318-24332 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to 24318-24332comprises a nucleotide sequence selected from SEQ ID NO 881, 869, 883,or 858. In certain such embodiments, a short antisense compound targetedto nucleotides 24318-24332 of SEQ ID NO: 11 is selected from Isis No147043, 147044, 147045, or 147046.

In certain embodiments, a target region is nucleotides 24486-24501 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 24486-24501 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to 24486-24501comprises a nucleotide sequence selected from SEQ ID NO 881, 869, 858,or 833. In certain such embodiments, a short antisense compound targetedto nucleotides 24486-24501 of SEQ ID NO: 11 is selected from Isis No147043, 147044, 147046, or 147047.

In certain embodiments, a target region is nucleotides 25065-25077 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 25065-25077 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to 25065-25077comprises a nucleotide sequence selected from SEQ ID NO 864 or 834. Incertain such embodiments, a short antisense compound targeted tonucleotides 25065-25077 of SEQ ID NO: 11 is selected from Isis No 147040or 147041.

In certain embodiments, a target region is nucleotides 25232-25245 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 25232-25245 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to 25232-25245comprises a nucleotide sequence selected from SEQ ID NO 850, 864, or834. In certain such embodiments, a short antisense compound targeted tonucleotides 25232-25245 of SEQ ID NO: 11 is selected from Isis No147039, 147040, or 147041.

In certain embodiments, a target region is nucleotides 25508-25523 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 25508-25523 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to 25508-25523comprises a nucleotide sequence selected from SEQ ID NO 839 or 879. Incertain such embodiments, a short antisense compound targeted tonucleotides 25508-25523 of SEQ ID NO: 11 is selected from Isis No 147057or 147061.

In certain embodiments, a target region is nucleotides 25676-28890 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 25676-28890 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to 25676-28890comprises a nucleotide sequence selected from SEQ ID NO 839, 860, or878. In certain such embodiments, a short antisense compound targeted tonucleotides 25676-28890 of SEQ ID NO: 11 is selected from Isis No147057, 147069, or 147070.

In certain embodiments, a target region is nucleotides 33056-33069 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 33056-33069 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to 33056-33069comprises a nucleotide sequence selected from SEQ ID NO 860, 878, or856. In certain such embodiments, a short antisense compound targeted tonucleotides 33056-33069 of SEQ ID NO: 11 is selected from Isis No147069, 147070, or 147071.

In certain embodiments, a target region is nucleotides 33205-33217 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 33205-33217 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted to 33205-33217comprises a nucleotide sequence selected from SEQ ID NO 878 or 856. Incertain such embodiments, a short antisense compound targeted tonucleotides 33205-33217 of SEQ ID NO: 11 is selected from Isis No 14707or 147071.

In certain embodiments, a target region is nucleotides 33318-33334 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 33318-33334 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted to 33318-33334comprises a nucleotide sequence selected from SEQ ID NO 858, 854, or875. In certain such embodiments, a short antisense compound targeted tonucleotides 33318-33334 of SEQ ID NO: 11 is selected from Isis No147046, 147049, or 147051.

In certain embodiments, a target region is nucleotides 33466-33482 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 33466-33482 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 33466-33482 comprises anucleotide sequence selected from SEQ ID NO 858, 833, or 875. In certainsuch embodiments, a short antisense compound targeted to nucleotides33466-33482 of SEQ ID NO: 11 is selected from Isis No 147046, 147047, or147051.

In certain embodiments, a target region is nucleotides 33640-33656 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 33640-33656 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 33640-33656 comprises anucleotide sequence selected from SEQ ID NO 858 or 875. In certain suchembodiments, a short antisense compound targeted to nucleotides33640-33656 of SEQ ID NO: 11 is selected from Isis No 147046 or 147051.

In certain embodiments, a target region is nucleotides 33788-33804 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 33788-33804 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 33788-33804 comprises anucleotide sequence selected from SEQ ID NO 858 or 875. In certain suchembodiments, a short antisense compound targeted to nucleotides33788-33804 of SEQ ID NO: 11 is selected from Isis No 147046 or 147051.

In certain embodiments, a target region is nucleotides 35437-35449 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 35437-35449 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 35437-35449 comprises anucleotide sequence selected from SEQ ID NO 840 or 861. In certain suchembodiments, a short antisense compound targeted to nucleotides35437-35449 of SEQ ID NO: 11 is selected from Isis No 147059 or 147060.

In certain embodiments, a target region is nucleotides 40353-40373 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 40353-40373 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 40353-40373 comprises anucleotide sequence selected from SEQ ID NO 879 or 881. In certain suchembodiments, a short antisense compound targeted to nucleotides40353-40373 of SEQ ID NO: 11 is selected from Isis No 147061 or 147043.

In certain embodiments, a target region is nucleotides 42527-42541 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 42527-42541 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 42527-42541 comprises anucleotide sequence selected from SEQ ID NO 885, 870, or 844. In certainsuch embodiments, a short antisense compound targeted to nucleotides42527-42541 of SEQ ID NO: 11 is selected from Isis No 147031, 147032, or147034.

In certain embodiments, a target region is nucleotides 42675-42689 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 42675-42689 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 42675-42689 comprises anucleotide sequence selected from SEQ ID NO 885, 870, 836, or 844. Incertain such embodiments, a short antisense compound targeted tonucleotides 42675-42689 of SEQ ID NO: 11 is selected from Isis No147031, 147032, 147033, or 147034.

In certain embodiments, a target region is nucleotides 46313-46328 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 46313-46328 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 46313-46328 comprises anucleotide sequence selected from SEQ ID NO 839, 830, 840, or 879. Incertain such embodiments, a short antisense compound targeted tonucleotides 46313-46328 of SEQ ID NO: 11 is selected from Isis No147057, 147058, 147059, or 147061.

In certain embodiments, a target region is nucleotides 46461-46476 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 46461-46476 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 46461-46476 comprises anucleotide sequence selected from SEQ ID NO 839, 840, or 879. In certainsuch embodiments, a short antisense compound targeted to nucleotides46461-46476 of SEQ ID NO: 11 is selected from Isis No 147057, 147059, or147061.

In certain embodiments, a target region is nucleotides 48369-48381 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 48369-48381 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 48369-48381 comprises anucleotide sequence selected from SEQ ID NO 842 or 845. In certain suchembodiments, a short antisense compound targeted to nucleotides48369-48381 of SEQ ID NO: 11 is selected from Isis No 147073 or 147074.

In certain embodiments, a target region is nucleotides 48714-48726 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 48714-48726 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 48714-48726 comprises anucleotide sequence selected from SEQ ID NO 843 or 846. In certain suchembodiments, a short antisense compound targeted to nucleotides48714-48726 of SEQ ID NO: 11 is selected from Isis No 147027 or 147028.

In certain embodiments, a target region is nucleotides 49050-49062 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 49050-49062 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 49050-49062 ofcomprises a nucleotide sequence selected from SEQ ID NO 876 or 838. Incertain such embodiments, a short antisense compound targeted tonucleotides 4905049062 of SEQ ID NO: 11 is selected from Isis No 147067or 147068.

In certain embodiments, a target region is nucleotides 49672-49684 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 4967249684 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 4967249684 of comprisesa nucleotide sequence selected from SEQ ID NO 842 or 845. In certainsuch embodiments, a short antisense compound targeted to nucleotides49672-49684 of SEQ ID NO: 11 is selected from Isis No 147073 or 147074.

In certain embodiments, a target region is nucleotides 52292-52304 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 52292-52304 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 52292-52304 ofcomprises a nucleotide sequence selected from SEQ ID NO 849 or 863. Incertain such embodiments, a short antisense compound targeted tonucleotides 52292-52304 of SEQ ID NO: 1 is selected from Isis No 147036or 147037.

In certain embodiments, a target region is nucleotides 52438-52450 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 52438-52450 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 52438-52450 ofcomprises a nucleotide sequence selected from SEQ ID NO 849 or 863. Incertain such embodiments, a short antisense compound targeted tonucleotides 52438-52450 of SEQ ID NO: 11 is selected from Isis No 147036or 147037.

In certain embodiments, a target region is nucleotides 53445-53458 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 53445-53458 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 53445-53458 ofcomprises a nucleotide sequence selected from SEQ ID NO 866, 881, or869. In certain such embodiments, a short antisense compound targeted tonucleotides 53445-53458 of SEQ ID NO: 11 is selected from Isis No147042, 147043, or 147044.

In certain embodiments, a target region is nucleotides 53591-53604 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 53591-53604 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 53591-53604 ofcomprises a nucleotide sequence selected from SEQ ID NO 866, 874, 881,885, or 869. In certain such embodiments, a short antisense compoundtargeted to nucleotides 53591-53604 of SEQ ID NO: 11 is selected fromIsis No 147042, 147030, 147043, 147031, or 147044.

In certain embodiments, a target region is nucleotides 53738-53750 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 53738-53750 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 53738-53750 ofcomprises a nucleotide sequence selected from SEQ ID NO 874 or 885. Incertain such embodiments, a short antisense compound targeted tonucleotides 53738-53750 of SEQ ID NO: 11 is selected from Isis No 147030or 147031.

In certain embodiments, a target region is nucleotides 53783-53795 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 53783-53795 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 53783-53795 ofcomprises a nucleotide sequence selected from SEQ ID NO 864 or 834. Incertain such embodiments, a short antisense compound targeted tonucleotides 53783-53795 of SEQ ID NO: 11 is selected from Isis No 147040or 147041.

In certain embodiments, a target region is nucleotides 55008-55020 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 55008-55020 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 55008-55020 ofcomprises a nucleotide sequence selected from SEQ ID NO 866 or 881. Incertain such embodiments, a short antisense compound targeted tonucleotides 55008-55020 of SEQ ID NO: 11 is selected from Isis No 147042or 147043.

In certain embodiments, a target region is nucleotides 55154-55166 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 55154-55166 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 55154-55166 ofcomprises a nucleotide sequence selected from SEQ ID NO 866 or 881. Incertain such embodiments, a short antisense compound targeted tonucleotides 55154-55166 of SEQ ID NO: 11 is selected from Isis No 147042or 147043.

In certain embodiments, a target region is nucleotides 55682-55695 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 55682-55695 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 55682-55695 ofcomprises a nucleotide sequence selected from SEQ ID NO 877 or 882. Incertain such embodiments, a short antisense compound targeted tonucleotides 55682-55695 of SEQ ID NO: 11 is selected from Isis No 147019or 147021.

In certain embodiments, a target region is nucleotides 56275-56293 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 56275-56293 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 56275-56293 ofcomprises a nucleotide sequence selected from SEQ ID NO 871, 884, 887,830, 840, 861, or 879. In certain such embodiments, a short antisensecompound targeted to nucleotides 56275-56293 of SEQ ID NO: 11 isselected from Isis No 147054, 147055, 147056, 147058, 147059, 147060, or147061.

In certain embodiments, a target region is nucleotides 56418-56439 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 56418-56439 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 56418-56439 ofcomprises a nucleotide sequence selected from SEQ ID NO 875, 829, 871,884, 887, 839, 830, or 879. In certain such embodiments, a shortantisense compound targeted to nucleotides 56418-56439 of SEQ ID NO: 11is selected from Isis No 147051, 147053, 147054, 147055, 147056, 147057,147058, or 147061.

In certain embodiments, a target region is nucleotides 57264-57276 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 57264-57276 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 57264-57276 ofcomprises a nucleotide sequence selected from SEQ ID NO 883 or 858. Incertain such embodiments, a short antisense compound targeted tonucleotides 57264-57276 of SEQ ID NO: 11 is selected from Isis No 147045or 147046.

In certain embodiments, a target region is nucleotides 61276-61293 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 61276-61293 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 61276-61293 ofcomprises a nucleotide sequence selected from SEQ ID NO 856, 847, 849,863, 855, 850, or 864. In certain such embodiments, a short antisensecompound targeted to nucleotides 61276-61293 of SEQ ID NO: 11 isselected from Isis No 147071, 147035, 147036, 147037, 147038, 147039, or147040.

In certain embodiments, a target region is nucleotides 61257-61320 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 61257-61320 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 61257-61320 ofcomprises a nucleotide sequence selected from SEQ ID NO 881, 856, 847,849, 863, 855, 850, 864, or 886. In certain such embodiments, a shortantisense compound targeted to nucleotides 61257-61320 of SEQ ID NO: 11is selected from Isis No 147043, 147071, 147035, 147036, 147037, 147038,147039, 147040, or 147071.

In certain embodiments, a target region is nucleotides 61422-61439 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 61422-61439 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 61422-61439 ofcomprises a nucleotide sequence selected from SEQ ID NO 844, 847, 849,863, 855, or 864. In certain such embodiments, a short antisensecompound targeted to nucleotides 61422-61439 of SEQ ID NO: 11 isselected from Isis No 147034, 147035, 147036, 147037, 147038, or 147040.

In certain embodiments, a target region is nucleotides 61422-61466 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 61422-61466 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 61422-61466 ofcomprises a nucleotide sequence selected from SEQ ID NO 844, 847, 849,863, 855, 864, or 856. In certain such embodiments, a short antisensecompound targeted to nucleotides 61422-61466 of SEQ ID NO: 11 isselected from Isis No 147034, 147035, 147036, 147037, 147038, 147040, or147071.

In certain embodiments, a target region is nucleotides 63065-63078 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 63065-63078 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 63065-63078 ofcomprises a nucleotide sequence selected from SEQ ID NO 851 or 838. Incertain such embodiments, a short antisense compound targeted tonucleotides 63065-63078 of SEQ ID NO: 11 is selected from Isis No 147066or 147068.

In certain embodiments, a target region is nucleotides 63207-63222 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 63207-63222 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 63207-63222 ofcomprises a nucleotide sequence selected from SEQ ID NO 841 or 851. Incertain such embodiments, a short antisense compound targeted tonucleotides 63207-63222 of SEQ ID NO: 11 is selected from Isis No 147062or 147066.

In certain embodiments, a target region is nucleotides 64538-64550 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 64538-64550 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 64538-64550 ofcomprises a nucleotide sequence selected from SEQ ID NO 849 or 863. Incertain such embodiments, a short antisense compound targeted tonucleotides 64538-64550 of SEQ ID NO: 11 is selected from Isis No 147036or 147037.

In certain embodiments, a target region is nucleotides 64864-64876 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 64864-64876 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 64864-64876 ofcomprises a nucleotide sequence selected from SEQ ID NO 851 or 876. Incertain such embodiments, a short antisense compound targeted tonucleotides 64864-64876 of SEQ ID NO: 11 is selected from Isis No 147066or 147067.

In certain embodiments, a target region is nucleotides 65010-65028 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 65010-65028 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 65010-65028 ofcomprises a nucleotide sequence selected from SEQ ID NO 851, 876, or883. In certain such embodiments, a short antisense compound targeted tonucleotides 65010-65028 of SEQ ID NO: 11 is selected from Isis No147066, 147067, or 147045.

In certain embodiments, a target region is nucleotides 65163-65175 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 65163-65175 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 65163-65175 ofcomprises a nucleotide sequence selected from SEQ ID NO 883 or 858. Incertain such embodiments, a short antisense compound targeted tonucleotides 65163-65175 of SEQ ID NO: 11 is selected from Isis No 147045or 147046.

In certain embodiments, a target region is nucleotides 65408-65422 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 65408-65422 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 65408-65422 ofcomprises a nucleotide sequence selected from SEQ ID NO 883 or 856. Incertain such embodiments, a short antisense compound targeted tonucleotides 65408-65422 of SEQ ID NO: 11 is selected from Isis No 147068or 147071.

In certain embodiments, a target region is nucleotides 65549-65568 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 65549-65568 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 65549-65568 ofcomprises a nucleotide sequence selected from SEQ ID NO 860, 838, or856. In certain such embodiments, a short antisense compound targeted tonucleotides 65549-65568 of SEQ ID NO: 11 is selected from Isis No147069, 147068, or 147071.

In certain embodiments, a target region is nucleotides 67741-67754 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 67741-67754 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 67741-67754 ofcomprises a nucleotide sequence selected from SEQ ID NO 848, 874, or885. In certain such embodiments, a short antisense compound targeted tonucleotides 67741-67754 of SEQ ID NO: 11 is selected from Isis No147029, 147030, or 147031.

In certain embodiments, a target region is nucleotides 67886-67900 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 67886-67900 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 67886-67900 ofcomprises a nucleotide sequence selected from SEQ ID NO 846, 848, 874,or 885. In certain such embodiments, a short antisense compound targetedto nucleotides 67886-67900 of SEQ ID NO: 11 is selected from Isis No147028, 147029, 147030, or 147031.

In certain embodiments, a target region is nucleotides 68867-68880 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 68867-68880 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 68867-68880 ofcomprises a nucleotide sequence selected from SEQ ID NO 881, 869, or883. In certain such embodiments, a short antisense compound targeted tonucleotides 68867-68880 of SEQ ID NO: 11 is selected from Isis No147043, 147044, or 147045.

In certain embodiments, a target region is nucleotides 69013-69532 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 69013-69532 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 69013-69532 ofcomprises a nucleotide sequence selected from SEQ ID NO 881, 869, 883,858, 856, 832, or 842. In certain such embodiments, a short antisensecompound targeted to nucleotides 69013-69532 of SEQ ID NO: 11 isselected from Isis No 147043, 147044, 147045, 147046, 147071, 147072, or147073.

In certain embodiments, a target region is nucleotides 69665-69880 ofSEQ ID NO: 1. In certain embodiments, a short antisense compound istargeted to nucleotides 69665-69880 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 69665-69880 ofcomprises a nucleotide sequence selected from SEQ ID NO 856, 832, 842,845, or 851. In certain such embodiments, a short antisense compoundtargeted to nucleotides 69665-69880 of SEQ ID NO: 11 is selected fromIsis No 147071, 147072, 147073, 147074, or 147066.

In certain embodiments, a target region is nucleotides 70611-70630 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 70611-70630 of SEQ ID NO: 1. In certain suchembodiments, a short antisense compound targeted 70611-70630 ofcomprises a nucleotide sequence selected from SEQ ID NO 859, 841, 862,880, 857, or 851. In certain such embodiments, a short antisensecompound targeted to nucleotides 70611-70630 of SEQ ID NO: 11 isselected from Isis No 147023, 147062, 147063, 147064, 147065, or 147066.

In certain embodiments, a target region is nucleotides 70762-70776 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 70762-70776 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 70762-70776 ofcomprises a nucleotide sequence selected from SEQ ID NO 862, 880, 857,or 851. In certain such embodiments, a short antisense compound targetedto nucleotides 70762-70776 of SEQ ID NO: 11 is selected from Isis No147063, 147064, 147065, or 147066.

In certain embodiments, a target region is nucleotides 70998-71010 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 70998-71010 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 70998-71010 ofcomprises a nucleotide sequence selected from SEQ ID NO 832 or 842. Incertain such embodiments, a short antisense compound targeted tonucleotides 70998-71010 of SEQ ID NO: 11 is selected from Isis No 147072or 147073.

In certain embodiments, a target region is nucleotides 71144-714364 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 71144-714364 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 71144-714364 ofcomprises a nucleotide sequence selected from SEQ ID NO 832, 842, 845,863, 855, or 850. In certain such embodiments, a short antisensecompound targeted to nucleotides 71144-714364 of SEQ ID NO: 11 isselected from Isis No 147072, 147073, 147074, 147037, 147038, or 147039.

In certain embodiments, a target region is nucleotides 71497-71652 ofSEQ ID NO: 11. In certain embodiments, a short antisense compound istargeted to nucleotides 71497-71652 of SEQ ID NO: 11. In certain suchembodiments, a short antisense compound targeted 71497-71652 ofcomprises a nucleotide sequence selected from SEQ ID NO 863, 855, 850,or 879. In certain such embodiments, a short antisense compound targetedto nucleotides 71497-71652 of SEQ ID NO: 11 is selected from Isis No147037, 147038, 147039, or 147061.

In certain embodiments, short antisense compounds targeted to a PTP1Bnucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14,more preferably 10 to 14 nucleotides in length. In certain embodiments,short antisense compounds targeted to a PTP1B nucleic acid are 9 to 14nucleotides in length. In certain embodiments, short antisense compoundstargeted to a PTP1B nucleic acid are 10 to 14 nucleotides in length. Incertain embodiments, such short antisense compounds are short antisenseoligonucleotides.

In certain embodiments, short antisense compounds targeted to a PTP1Bnucleic acid are short gapmers. In certain such embodiments, shortgapmers targeted to a PTP1B nucleic acid comprise at least one highaffinity modification in one or more wings of the compound. In certainembodiments, short antisense compounds targeted to a PTP1B nucleic acidcomprise 1 to 3 high-affinity modifications in each wing. In certainsuch embodiments, the nucleosides or nucleotides of the wing comprise a2′ modification. In certain such embodiments, the monomers of the wingare BNA's. In certain such embodiments, the monomers of the wing areselected from α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, β-D-Methyleneoxy(4′-CH₂—O-2′) BNA, Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, Aminooxy(4′-CH₂—O—N(R)-2′) BNA and Oxyamino (4′-CH₂—N(R)—O-2′) BNA. In certainembodiments, the monomers of a wing comprise a substituent at the 2′position selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H or substituted or unsubstitutedC₁-C₁₀ alkyl. In certain embodiments, the monomers of a wing are 2′MOEnucleotides.

In certain embodiments, short antisense compounds targeted to a PTP1Bnucleic acid comprise a gap between the 5′ wing and the 3′ wing. Incertain embodiments the gap comprises five, six, seven, eight, nine,ten, eleven, twelve, thirteen, or fourteen monomers. In certainembodiments, the monomers of the gap are unmodifieddeoxyribonucleotides. In certain embodiments, the monomers of the gapare unmodified ribonucleotides. In certain embodiments, gapmodifications (if any) gap result in an antisense compound that, whenbound to its target nucleic acid, supports cleavage by an RNase,including, but not limited to, RNase H.

In certain embodiments, short antisense compounds targeted to a PTP1Bnucleic acid have uniform monomeric linkages. In certain suchembodiments, those linkages are all phosphorothioate linkages. Incertain embodiments, the linkages are all phosphodiester linkages. Incertain embodiments, short antisense compounds targeted to a PTP1Bnucleic acid have mixed backbones.

In certain embodiments, short antisense compounds targeted to a PTP1Bnucleic acid are 8 monomers in length. In certain embodiments, shortantisense compounds targeted to a PTP1B nucleic acid are 9 monomers inlength. In certain embodiments, short antisense compounds targeted to aPTP1B nucleic acid are 10 monomers in length. In certain embodiments,short antisense compounds targeted to a PTP1B nucleic acid are 11monomers in length. In certain embodiments, short antisense compoundstargeted to a PTP1B nucleic acid are monomers in length. In certainembodiments, short antisense compounds targeted to a PTP1B nucleic acidare 13 monomers in length. In certain embodiments, short antisensecompounds targeted to a PTP1B nucleic acid are 14 monomers in length. Incertain embodiments, short antisense compounds targeted to a PTP1Bnucleic acid are 15 monomers in length. In certain embodiments, shortantisense compounds targeted to a PTP1B nucleic acid are 16 monomers inlength. In certain embodiments, short antisense compounds targeted to aPTP1B nucleic acid comprise 9 to 15 monomers. In certain embodiments,short antisense compounds targeted to a PTP1B nucleic acid comprise 10to 15 monomers. In certain embodiments, short antisense compoundstargeted to a PTP1B nucleic acid comprise 12 to 14 monomers. In certainembodiments, short antisense compounds targeted to a PTP1B nucleic acidcomprise 12 to 14 nucleotides or nucleosides.

In certain embodiments, the invention provides methods of modulatingexpression of PTP1B. In certain embodiments, such methods comprise useof one or more short antisense compound targeted to a PTP1B nucleicacid, wherein the short antisense compound targeted to a PTP1B nucleicacid is from about 8 to about 16, preferably 9 to 15, more preferably 9to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16linked monomers). One of ordinary skill in the art will appreciate thatthis comprehends methods of modulating expression of PTP1B using one ormore short antisense compounds targeted to a PTP1B nucleic acid of 8, 9,10, 11, 12, 13, 14, 15 or 16 monomers.

In certain embodiments, methods of modulating PTP1B comprise use of ashort antisense compound targeted to a PTP1B nucleic acid that is 8monomers in length. In certain embodiments, methods of modulating PTP1Bcomprise use of a short antisense compound targeted to a PTP1B nucleicacid that is 9 monomers in length. In certain embodiments, methods ofmodulating PTP1B comprise use of a short antisense compound targeted toa PTP1B nucleic acid that is 10 monomers in length. In certainembodiments, methods of modulating PTP1B comprise use of a shortantisense compound targeted to a PTP1B nucleic acid that is 11 monomersin length. In certain embodiments, methods of modulating PTP1B compriseuse of a short antisense compound targeted to a PTP1B nucleic acid thatis 12 monomers in length. In certain embodiments, methods of modulatingPTP1B comprise use of a short antisense compound targeted to a PTP1Bnucleic acid that is 13 monomers in length. In certain embodiments,methods of modulating PTP1B comprise use of a short antisense compoundtargeted to a PTP1B nucleic acid that is 14 monomers in length. Incertain embodiments, methods of modulating PTP1B comprise use of a shortantisense compound targeted to a PTP1B nucleic acid that is 15 monomersin length. In certain embodiments, methods of modulating PTP1B compriseuse of a short antisense compound targeted to a PTP1B nucleic acid thatis 16 monomers in length.

In certain embodiments, methods of modulating expression of PTP1Bcomprise use of a short antisense compound targeted to a PTP1B nucleicacid comprising 9 to 15 monomers. In certain embodiments, methods ofmodulating expression of PTP1B comprise use of a short antisensecompound targeted to a PTP1B nucleic acid comprising 10 to 15 monomers.In certain embodiments, methods of modulating expression of PTP1Bcomprise use of a short antisense compound targeted to a PTP1B nucleicacid comprising 12 to 14 monomers. In certain embodiments, methods ofmodulating expression of PTP1B comprise use of a short antisensecompound targeted to a PTP1B nucleic acid comprising 12 or 14nucleotides or nucleosides.

10. PTEN

In certain embodiments, the invention provides short antisense compoundstargeted to a nucleic acid encoding PTEN. In certain embodiments, suchcompounds are used to modulate PTEN expression if cells. In certain suchembodiments, short antisense compounds targeted to a PTEN nucleic acidare administered to an animal. In certain embodiments, short antisensecompounds targeted to a PTEN nucleic acid are useful for studying PTEN,for studying certain nucleases and/or for assessing antisense activity.In certain such embodiments, short antisense compounds targeted to PTENnucleic acids are useful for assessing certain motifs and/or chemicalmodifications. In certain embodiments, administration of a shortantisense compound targeted to PTEN nucleic acid to an animal results ina measurable phenotypic change.

The short antisense compounds targeting PTEN may have any one or moreproperties or characteristics of the short antisense compounds generallydescribed herein. In certain embodiments, short antisense compoundstargeting a PTP1B nucleic acid have a motif (wing-deoxy gap-wing)selected from 1-12-1, 1-1-10-2, 2-10-1-1, 3-10-3, 2-10-3, 2-10-2,1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or 1-6-1, more preferably1-10-1, 2-10-2, 3-10-3, and 1-9-2.

Certain Short Antisense Compounds Targeted to a PTEN Nucleic Acid

In certain embodiments, short antisense compounds are targeted to a PTENnucleic acid having the sequence of GENBANK® Accession No. NM 000314.4,incorporated herein as SEQ ID NO: 14. In certain embodiments, shortantisense compounds are targeted to a PTEN nucleic acid having thesequence of nucleotides 8063255 to 8167140 of the sequence of GENBANK®Accession No. NT 033890.3, incorporated herein as SEQ ID NO: 15. Incertain such embodiments, a short antisense compound targeted to SEQ IDNO: 14 is at least 90% complementary to SEQ ID NO: 14. In certain suchembodiments, a short antisense compound targeted to SEQ ID NO: 14 is atleast 95% complementary to SEQ ID NO: 14. In certain such embodiments, ashort antisense compound targeted to SEQ ID NO: 15 is 100% complementaryto SEQ ID NO: 15. In certain such embodiments, a short antisensecompound targeted to SEQ ID NO: 15 is at least 90% complementary to SEQID NO: 15. In certain such embodiments, a short antisense compoundtargeted to SEQ ID NO: 15 is at least 95% complementary to SEQ ID NO:15. In certain such embodiments, a short antisense compound targeted toSEQ ID NO: 15 is 100% complementary to SEQ ID NO: 15.

In certain embodiments, a short antisense compound targeted to SEQ IDNO: 14 comprises a nucleotide sequence selected from the nucleotidesequences set forth in Tables 20 and 21. In certain embodiments, a shortantisense compound targeted to SEQ ID NO: 15 comprises a nucleotidesequence selected from the nucleotide sequences set forth in Tables 22and 23.

Each nucleotide sequence set forth in Tables 20, 21, 22, and 23 isindependent of any modification to a sugar moiety, an internucleosidelinkage, or a nucleobase. As such, short antisense compounds comprisinga nucleotide sequence as set forth in Tables 20, 21, 22, and 23 maycomprise, independently, one or more modifications to a sugar moiety, aninternucleoside linkage, or a nucleobase. Antisense compounds describedby Isis Number (Isis NO.) indicate a combination of nucleobase sequenceand one or more modifications to a sugar moiety, an internucleosidelinkage, or a nucleobase.

Table 20 illustrates short antisense compounds that are 100%complementary to SEQ ID NO: 14. Table 22 illustrates short antisensecompounds that are 100% complementary to SEQ ID NO: 15. The columnlabeled ‘gapmer motif’ indicates the wing-gap-wing motif of each shortantisense compounds. The gap segment comprises 2′-deoxynucleotides andeach nucleotide of each wing segment comprises a 2′-modified sugar. Theparticular 2′-modified sugar is also indicated in the ‘gapmer motif’column. For example, ‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where agap segment of ten 2′-deoxynucleotides is flanked by wing segments oftwo nucleotides, where the nucleotides of the wing segments are 2′-MOEnucleotides. Internucleoside linkages are phosphorothioate. The shortantisense compounds comprise 5-methylcytidine in place of unmodifiedcytosine, unless “unmodified cytosine” is listed in the gapmer motifcolumn, in which case the indicated cytosines are unmodified cytosines.For example, “5-mC in gap only” indicates that the gap segment has5-methylcytosines, while the wing segments have unmodified cytosines.

The 2′-modified nucleotides and abbreviations include: 2′-O-methoxyethyl(MOE); 2′-O-methyl (OMe); 2′-O-(2,2,3,3,3-pentafluoropropyl) (PentaF);2′-O-[(2-methoxy)ethyl]-4′-thio (2′-MOE-4′-thio); (R)-CMOE-BNA. Asillustrated in Tables 20 and 22, a wing may comprise monomers comprisingmore than type of 2′ substituent. For example, 1-2-10-2 MOE/PentaF/MOEindicates one MOE-modified nucleotide, followed by two PentaF-modifiednucleotides, followed by a gap of ten deoxynucleotides, followed by twoPentaF-modified nucleotides. For example,1-1-10-22′-(butylacetomido)-palmitamide Methyleneoxy BNA/MethyleneoxyBNA indicates that the 5′-most nucleotide is2′-(butylacetomide)-palmitamide, the second nucleotide is a methyleneoxyBNA nucleotide, and the 3′ wing is methyleneoxy BNA. Unless otherwiseindicated, cytosines are 5-methylcytosines and internucleoside linkagesare phosphorothioate.

TABLE 20 Short Antisense Compounds Targeted to SEQ ID NO: 14 5′ 3′ SEQISIS Target Target ID No Site Site Sequence (5′-3′) Gapmer Motif NO390092 5530 5541 AGAATGAGACTT 1-10-1 MOE 1514 390091 5435 5446TGAGGCATTATC 1-10-1 MOE 1522 390090 5346 5357 AGAGTATCTGAA 1-10-1 MOE1227 390088 5162 5173 CACATTAACAGT 1-10-1 MOE 1511 390087 5126 5137GTGGCAACCACA 1-10-1 MOE 1501 390085 5031 5042 ATTTGATGCTGC 1-10-1 MOE1505 390084 4982 4993 CAAAGAATGGTG 1-10-1 MOE 1215 390082 4910 4921AGGACTTGGGAT 1-10-1 MOE 1503 390080 4833 4844 TGCTGCACATCC 1-10-1 MOE1150 392067 4832 4845 CTGCTGCACATCCA 2-10-2 Methyleneoxy BNA 1510Unmodified cytosines in gap 390078 4714 4725 CTTTCAGTCATA 1-10-1 MOE1520 390077 4693 4704 GTCAAATTCTAT 1-10-1 MOE 1252 390076 4599 4610TTCCAATGACTA 1-10-1 MOE 1506 390075 4576 4587 GTAAGCAAGGCT 1-10-1 MOE#N/A 390074 4533 4544 ACCCTCATTCAG 1-10-1 MOE 1513 390068 4191 4202GTAAATCCTAAG 1-10-1 MOE 1515 390064 4001 4012 ACCACAGCTAGT 1-10-1 MOE1498 390063 3977 3988 CACCAATAAGTT 1-10-1 MOE 1219 390058 3828 3839AGTAGTTGTACT 1-10-1 MOE 1192 390056 3793 3804 GGGCATATCAAA 1-10-1 MOE1521 390054 3705 3716 AACACTGCACAT 1-10-1 MOE 1493 390052 3623 3634GACAATTTCTAC 1-10-1 MOE 1492 390050 3503 3514 GTATTCAAGTAA 1-10-1 MOE1140 390049 3479 3490 GTTAATGACATT 1-10-1 MOE 1491 390047 3428 3439TGTGTAAGGTCA 1-10-1 MOE 1490 390041 3175 3186 TTAGCACTGGCC 1-10-1 MOE1489 398076 3171 3182 CACTGGCCTTGA 1-10-1 MOE 1488 398009 3170 3183GCACTGGCCTTGAT 2-10-2 MOE 1487 398075 3111 3122 AAATCATTGTCA 1-10-1 MOE1233 398008 3110 3123 TAAATCATTGTCAA 2-10-2 MOE 1486 398074 2913 2924GCACCAATATGC 1-10-1 MOE 1248 398007 2912 2925 AGCACCAATATGCT 2-10-2 MOE1247 398073 2681 2692 TTAGCCAACTGC 1-10-1 MOE 1485 398006 2680 2693CTTAGCCAACTGCA 2-10-2 MOE 1484 390033 2679 2690 AGCCAACTGCAA 1-10-1 MOE1483 398072 2671 2682 GCAAACTTATCT 1-10-1 MOE 1482 398005 2670 2683TGCAAACTTATCTG 2-10-2 MOE 1481 390030 2534 2545 TTTATAAAACTG 1-10-1 MOE1074 398071 2533 2544 TTATAAAACTGG 1-10-1 MOE 1480 398004 2532 2545TTTATAAAACTGGA 2-10-2 MOE 1479 390029 2510 2521 AAAGTGCCATCT 1-10-1 MOE1478 390028 2491 2502 TCCTAATTGAAT 1-10-1 MOE 1477 398070 2481 2492ATTTTAAATGTC 1-10-1 MOE 1476 398003 2480 2493 AATTTTAAATGTCC 2-10-2 MOE1475 390027 2455 2466 AGGTATATACAT 1-10-1 MOE 1206 398069 2451 2462ATATACATGACA 1-10-1 MOE 1474 398002 2450 2463 TATATACATGACAC 2-10-2 MOE1473 398068 2440 2451 ACAGCTACACAA 1-10-1 MOE 1472 398001 2439 2452CACAGCTACACAAC 2-10-2 MOE 1471 390026 2438 2449 AGCTACACAACC 1-10-1 MOE1470 390025 2406 2417 GTGTCAAAACCC 1-10-1 MOE 1211 398067 2405 2416TGTCAAAACCCT 1-10-1 MOE 1210 398000 2404 2417 GTGTCAAAACCCTG 2-10-2 MOE1469 398066 2372 2383 AGATTGGTCAGG 1-10-1 MOE 1468 397999 2371 2384AAGATTGGTCAGGA 2-10-2 MOE 1467 398065 2349 2360 GTTCCTATAACT 1-10-1 MOE1466 397998 2348 2361 TGTTCCTATAACTG 2-10-2 MOE 1465 398064 2331 2342CTGACACAATGT 1-10-1 MOE 1464 397997 2330 2343 TCTGACACAATGTC 2-10-2 MOE1463 398063 2321 2332 GTCCTATTGCCA 1-10-1 MOE 1205 397996 2320 2333TGTCCTATTGCCAT 2-10-2 MOE 1462 390022 2286 2297 CAGTTTATTCAA 1-10-1 MOE1142 336221 2230 2243 TCAGACTTTTGTAA 3-8-3 MOE 1461 336220 2224 2237TTTTGTAATTTGTG 3-8-3 MOE 1460 336219 2209 2222 ATGCTGATCTTCAT 3-8-3 MOE1459 390021 2203 2214 CTTCATCAAAAG 1-10-1 MOE 1458 336218 2201 2214CTTCATCAAAAGGT 3-8-3 MOE 1457 389779 2201 2212 TCATCAAAAGGT 1-9-2 MOE1176 389979 2201 2212 TCATCAAAAGGT 1-10-1 MOE 1176 397995 2200 2213TTCATCAAAAGGTT 2-10-2 MOE 1456 336217 2192 2205 AAGGTTCATTCTCT 3-8-3 MOE1455 390020 2183 2194 TCTGGATCAGAG 1-10-1 MOE 1149 336216 2182 2195CTCTGGATCAGAGT 3-8-3 MOE 1454 336215 2169 2182 TCAGTGGTGTCAGA 3-8-3 MOE1453 398062 2166 2177 GGTGTCAGAATA 1-10-1 MOE 1255 397994 2165 2178TGGTGTCAGAATAT 2-10-2 MOE 1452 390019 2163 2174 GTCAGAATATCT 1-10-1 MOE1173 336214 2157 2170 GAATATCTATAATG 3-8-3 MOE 1573 398061 2151 2162ATAATGATCAGG 1-10-1 MOE 1451 397993 2150 2163 TATAATGATCAGGT 2-10-2 MOE1450 336213 2146 2159 ATGATCAGGTTCAT 3-8-3 MOE 1449 389778 2144 2155TCAGGTTCATTG 1-9-2 MOE 1448 389978 2144 2155 TCAGGTTCATTG 1-10-1 MOE1448 398060 2137 2148 CATTGTCACTAA 1-10-1 MOE 1447 336212 2136 2149TCATTGTCACTAAC 3-8-3 MOE 1446 397992 2136 2149 TCATTGTCACTAAC 2-10-2 MOE1446 336211 2112 2125 ACAGAAGTTGAACT 3-8-3 MOE 1445 390017 2111 2122GAAGTTGAACTG 1-10-1 MOE 1444 398059 2108 2119 GTTGAACTGCTA 1-10-1 MOE1443 397991 2107 2120 AGTTGAACTGCTAG 2-10-2 MOE 1442 336210 2104 2117TGAACTGCTAGCCT 3-8-3 MOE 1441 335340 2104 2118 TTGAACTGCTAGCCT 1-10-4MOE 1440 335339 2103 2117 TGAACTGCTAGCCTC 1-10-4 MOE 1439 335338 21022116 GAACTGCTAGCCTCT 1-10-4 MOE 1438 335337 2101 2115 AACTGCTAGCCTCTG1-10-4 MOE 1437 335336 2100 2114 ACTGCTAGCCTCTGG 1-10-4 MOE 1436 3904302099 2111 GCTAGCCTCTGGA 1-10-2 MOE 1163 Unmodified cytosines 390431 20992111 GCTAGCCTCTGGA 1-10-2 MOE 1163 Unmodified cytosines C in wing 9-(aminoethoxy)phenoxazine 390432 2099 2111 GCTAGCCTCTGGA 1-10-2 MOE 1163390433 2099 2111 GCTAGCCTCTGGA 1-10-2 MOE 1163 Unmodified cytosines Nt 6is 9-(aminoethoxy)phenoxazine 390434 2099 2111 GCTAGCCTCTGGA 1-10-2 MOE1163 Unmodified cytosines Nt 7 is 9-(aminoethoxy)phenoxazine 390435 20992111 GCTAGCCTCTGGA 1-10-2 MOE 1163 Unmodified cytosines Nt 9 is9-(aminoethoxy)phenoxazine 335335 2099 2113 CTGCTAGCCTCTGGA 1-10-4 MOE1435 389777 2098 2109 TAGCCTCTGGAT 1-9-2 MOE 1434 389954 2098 2109TAGCCTCTGGAT 1-10-1 MOE 1434 335334 2098 2112 TGCTAGCCTCTGGAT 1-10-4 MOE1433 331429 2097 2110 CTAGCCTCTGGATT 2-10-2 MOE 1431 335349 2097 2110CTAGCCTCTGGATT 2-10-2 MOE 1431 335367 2097 2110 CTAGCCTCTGGATT 2-10-2Methyleneoxy BNA 1431 335378 2097 2110 CTAGCCTCTGGATT 2-10-2Methyleneoxy BNA 1431 392061 2097 2110 CTAGCCTCTGGATT 2-10-2Methyleneoxy BNA 1431 Unmodified cytosines in gap 383991 2097 2109TAGCCTCTGGATT 1-10-2 1432 2′-(acetylamino-butyl-acetamido)-cholesterol/MOE 383992 2097 2109 TAGCCTCTGGATT 1-10-2 14322′-(acetylamino-butyl-acetamido)- cholic acid/MOE 386970 2097 2109TAGCCTCTGGATT 1-10-2 MOE 1432 390578 2097 2109 TAGCCTCTGGATT 1-10-2 MOE1432 Unmodified cytosines Ts in wings are 2-thiothymines 390614 20972109 TAGCCTCTGGATT 1-10-2 PentaF 1432 335333 2097 2111 GCTAGCCTCTGGATT1-10-4 MOE 1430 386683 2097 2109 TAGCCTCTGGATT 1-10-22′-(butylacetamido)- 1432 palmitamide/MOE 371975 2096 2110CTAGCCTCTGGATTT 3-10-2 MOE 1429 335341 2096 2111 GCTAGCCTCTGGATTT 3-10-3MOE 1428 335350 2096 2111 GCTAGCCTCTGGATTT 3-10-3 MOE 1428 335368 20962111 GCTAGCCTCTGGATTT 3-10-3 Methyleneoxy BNA 1428 Phosphodiesterlinkages in wings 335379 2096 2111 GCTAGCCTCTGGATTT 3-10-3 MethyleneoxyBNA 1428 383739 2096 2111 GCTAGCCTCTGGATTT 3-10-3 MOE 14285-methylcytosine in gap 384071 2096 2111 GCTAGCCTCTGGATTT 3-10-3 OMe1428 5-methylcytosine in gap 384073 2096 2111 GCTAGCCTCTGGATTT 3-10-3Methyleneoxy BNA 1428 5-methylcytosine in gap 390576 2096 2111GCTAGCCTCTGGATTT 3-10-3 MOE 1428 5-methylcytosine in gap T's in wingsare 2-thiothymines 390580 2096 2111 GCTAGCCTCTGGATTT 3-10-3 MOE 1428Pyrimidines in wings are 5-thiazole Unmodified cytosines in gap 3905812096 2111 GCTAGCCTCTGGATTT 3-10-3 MOE 1428 Unmodified cytosines in gap391863 2096 2111 GCTAGCCTCTGGATTT 3-10-3 MOE 1428 Unmodified cytosines391864 2096 2111 GCTAGCCTCTGGATTT 3-10-3 Methyleneoxy BNA 1428Unmodified cytosines in gap 391865 2096 2111 GCTAGCCTCTGGATTT 3-10-3Methyleneoxy BNA 1428 Unmodified cytosines 375560 2096 2110CTAGCCTCTGGATTT 2-10-3 MOE 1429 391172 2096 2110 CTAGCCTCTGGATTT 2-10-2Methyleneoxy BNA 1429 Unmodified cytosines 391175 2096 2110CTAGCCTCTGGATTT 2-10-3 Methyleneoxy BNA 1429 391449 2096 2110CTAGCCTCTGGATTT 2-10-3 MOE 1429 Unmodified cytosines 392054 2096 2110CTAGCCTCTGGATTT 2-10-3 Methyleneoxy BNA 1429 Unmodified cytosines in gap392055 2096 2110 CTAGCCTCTGGATTT 2-10-3 MOE 1429 Unmodified cytosines ingap 362977 2096 2111 GCTAGCCTCTGGATTT 2-12-2 MOE 1428 386770 2096 2109TAGCCTCTGGATTT 1-11-2 MOE 1427 390577 2096 2109 TAGCCTCTGGATTT 1-10-3MOE 1427 Unmodified cytosines T's in wings are 2-thiothymines 3353322096 2110 CTAGCCTCTGGATTT 1-10-4 MOE 1429 390579 2096 2111GCTAGCCTCTGGATTT 1-1-1-10-3 MOE/4′-thio/2′-O-[(2- 1428methoxy)ethyl]-4′-thio/2′-O-[(2- methoxy)ethyl]-4′-thio Unmodifiedcytosines in wings Phosphorodiester linkage in wings 391173 2096 2110CTAGCCTCTGGATTT 2-10-3 (5′R)-5′-methyl- 1429 Methyleneoxy BNA Unmodifiedcytosines 391174 2096 2110 CTAGCCTCTGGATTT 2-10-3 (5′S)-5′-methyl- 1429Methyleneoxy BNA Unmodified cytosines 390607 2096 2111 GCTAGCCTCTGGATTT3-10-3 MOE/pentaF 1428 Unmodified cytosines in wing 390609 2096 2111GCTAGCCTCTGGATTT 3-10-2-1 MOE/MOE/pentaF 1428 Unmodified cytosines inwing 384072 2096 2111 GCTAGCCTCTGGATTT 1-2-10-3 MOE/pentaF/pentaF 1428Unmodified cytosines in wings 390606 2096 2111 GCTAGCCTCTGGATTT 1-2-10-3MOE/pentaF/pentaF 1428 Unmodified cytosines in wing 390608 2096 2111GCTAGCCTCTGGATTT 1-2-10-3 MOE/pentaF/pentaF 1428 Unmodified cytosines inwing 391869 2096 2111 GCTAGCCTCTGGATTT 1-2-10-3 Methyleneoxy BNA/(5′S)-1428 5′-methyl-Methyleneoxy BNA/ (5′S)-5′-methyl-Methyleneoxy BNAUnmodified cytosines 385036 2096 2111 GCTAGCCTCTGGATTT 1-2-10-3OMe/2′-O-methyl-4′- 1428 thio/2′-O-methyl-4′-thio Unmodified cytosinesin wing 385871 2096 2111 GCTAGCCTCTGGATTT 1-2-10-3 OMe/2′-O-[(2- 1428methoxy)ethyl]-4′-thio/2′-O-[(2- methoxy)ethyl]-4′-thio Unmodifiedcytosines in wing 386682 2096 2111 GCTAGCCTCTGGATTT 1-2-10-32′-(butylacetamido)- 1428 palmitamide/MOE/MOE 390582 2096 2111GCTAGCCTCTGGATTT 1-2-10-3 MOE/2′-O-[(2- 1428methoxy)ethyl]-4′-thio/2′-O-[(2- methoxy)ethyl]-4′-thio Unmodifiedcytosines in wings Phosphodiester linkage in wings 391868 2096 2111GCTAGCCTCTGGATTT 1-2-10-3 (5′R)-5′-methyl- 1428 MethyleneoxyBNA/Methyleneoxy BNA/(5′R)-5′-methyl- Methyleneoxy BNA Unmodifiedcytosines 336209 2095 2108 AGCCTCTGGATTTG 3-8-3 MOE 1425 335331 20952109 TAGCCTCTGGATTTG 1-10-4 MOE 1426 335376 2095 2109 TAGCCTCTGGATTTG1-10-4 Methyleneoxy BNA 1426 335377 2095 2109 TAGCCTCTGGATTTG 1-10-4Methyleneoxy BNA 1426 Phosphodiester in 3′ wing 335330 2094 2108AGCCTCTGGATTTGA 1-10-4 MOE 1424 336208 2079 2092 GGCTCCTCTACTGT 3-8-3MOE 1423 336207 2073 2086 TCTACTGTTTTTGT 3-8-3 MOE 1422 336206 2047 2060CACCTTAAAATTTG 3-8-3 MOE 1518 389776 2046 2057 CTTAAAATTTGG 1-9-2 MOE1421 389977 2046 2057 CTTAAAATTTGG 1-10-1 MOE 1421 397990 2045 2058CCTTAAAATTTGGA 2-10-2 MOE 1420 336205 2043 2056 TTAAATTTGGAGA 3-8-3 MOE1419 398058 2029 2040 AGTATCGGTTGG 1-10-1 MOE 1418 336204 2028 2041AAGTATCGGTTGGC 3-8-3 MOE 1417 397989 2028 2041 AAGTATCGGTTGGC 2-10-2 MOE1417 336203 2002 2015 TGCTTTGTCAAGAT 3-8-3 MOE 1416 389775 2002 2013CTTTGTCAAGAT 1-9-2 MOE 1177 389976 2002 2013 CTTTGTCAAGAT 1-10-1 MOE1177 397988 2001 2014 GCTTTGTCAAGATC 2-10-2 MOE 1415 336202 1959 1972TCCTTGTCATTATC 3-8-3 MOE 1414 389774 1945 1956 CACGCTCTATAC 1-9-2 MOE1413 389975 1945 1956 CACGCTCTATAC 1-10-1 MOE 1413 336201 1944 1957GCACGCTCTATACT 3-8-3 MOE 1412 336200 1929 1942 CAAATGCTATCGAT 3-8-3 MOE1411 389773 1904 1915 AGACTTCCATTT 1-9-2 MOE 1410 389974 1904 1915AGACTTCCATTT 1-10-1 MOE 1410 336199 1902 1915 AGACTTCCATTTTC 3-8-3 MOE1409 336198 1884 1897 TTTTCTGAGGTTTC 3-8-3 MOE 1408 398057 1878 1889GGTTTCCTCTGG 1-10-1 MOE 1407 397987 1877 1890 AGGTTTCCTCTGGT 2-10-2 MOE1406 336197 1873 1886 TTCCTCTGGTCCTG 3-8-3 MOE 1405 390015 1868 1879GGTCCTGGTATG 1-10-1 MOE 1404 398056 1865 1876 CCTGGTATGAAG 1-10-1 MOE1403 336196 1864 1877 TCCTGGTATGAAGA 3-8-3 MOE 1402 397986 1864 1877TCCTGGTATGAAGA 2-10-2 MOE 1402 398055 1849 1860 TATTTACCCAAA 1-10-1 MOE1401 397985 1848 1861 GTATTTACCCAAAA 2-10-2 MOE 1400 336195 1847 1860TATTTACCCAAAAG 3-8-3 MOE 1399 389772 1846 1857 TTACCCAAAAGT 1-9-2 MOE1398 389973 1846 1857 TTACCCAAAAGT 1-10-1 MOE 1398 336194 1838 1851AAAAGTGAAACATT 3-8-3 MOE 1145 398054 1836 1847 GTGAAACATTTT 1-10-1 MOE1144 397984 1835 1848 AGTGAAACATTTTG 2-10-2 MOE 1397 336193 1828 1841CATTTTGTCCTTTT 3-8-3 MOE 1182 336192 1810 1823 CATCTTGTTCTGTT 3-8-3 MOE1396 336191 1800 1813 TGTTTGTGGAAGAA 3-8-3 MOE 1395 398053 1796 1807TGGAAGAACTCT 1-10-1 MOE 1394 397983 1795 1808 GTGGAAGAACTCTA 2-10-2 MOE1393 389771 1794 1805 GAAGAACTCTAC 1-9-2 MOE 1392 389972 1794 1805GAAGAACTCTAC 1-10-1 MOE 1392 336190 1789 1802 GAACTCTACTTTGA 3-8-3 MOE1391 336189 1773 1786 TCACCACACACAGG 3-8-3 MOE 1390 336188 1754 1767GCTGAGGGAACTCA 3-8-3 MOE 1389 398052 1751 1762 GGGAACTCAAAG 1-10-1 MOE1388 389770 1750 1761 GGAACTCAAAGT 1-9-2 MOE 1386 389971 1750 1761GGAACTCAAAGT 1-10-1 MOE 1386 397982 1750 1763 AGGGAACTCAAAGT 2-10-2 MOE1387 336187 1747 1760 GAACTCAAAGTACA 3-8-3 MOE 1385 390012 1745 1756TCAAAGTACATG 1-10-1 MOE 1384 336186 1688 1701 TCTTCACCTTTAGC 3-8-3 MOE1383 398051 1684 1695 CCTTTAGCTGGC 1-10-1 MOE 1220 397981 1683 1696ACCTTTAGCTGGCA 2-10-2 MOE 1382 336185 1677 1690 AGCTGGCAGACCAC 3-8-3 MOE1381 389769 1676 1687 TGGCAGACCACA 1-9-2 MOE 1249 389970 1676 1687TGGCAGACCACA 1-10-1 MOE 1249 392060 1675 1688 CTGGCAGACCACAA 2-10-2Methyleneoxy BNA 1380 Unmodified cytosines in gap 398050 1672 1683AGACCACAAACT 1-10-1 MOE 1379 397980 1671 1684 CAGACCACAAACTG 2-10-2 MOE1378 390011 1658 1669 GGATTGCAAGTT 1-10-1 MOE 1238 336184 1655 1668GATTGCAAGTTCCG 3-8-3 MOE 1508 336183 1644 1657 CCGCCACTGAACAT 3-8-3 MOE1377 390010 1643 1654 CCACTGAACATT 1-10-1 MOE 1240 398049 1641 1652ACTGAACATTGG 1-10-1 MOE 1376 397979 1640 1653 CACTGAACATTGGA 2-10-2 MOE1375 336182 1633 1646 CATTGGAATAGTTT 3-8-3 MOE 1374 389768 1630 1641GAATAGTTTCAA 1-9-2 MOE 1373 389969 1630 1641 GAATAGTTTCAA 1-10-1 MOE1373 398048 1626 1637 AGTTTCAAACAT 1-10-1 MOE 1372 397978 1625 1638TAGTTTCAAACATC 2-10-2 MOE 1371 336181 1623 1636 GTTTCAAACATCAT 3-8-3 MOE1370 398047 1614 1625 CATCTTGTGAAA 1-10-1 MOE 1369 336180 1613 1626TCATCTTGTGAAAC 3-8-3 MOE 1368 390009 1613 1624 ATCTTGTGAAAC 1-10-1 MOE1175 397977 1613 1626 TCATCTTGTGAAAC 2-10-2 MOE 1368 390007 1563 1574CAGGTAGCTATA 1-10-1 MOE 1367 336179 1561 1574 CAGGTAGCTATAAT 3-8-3 MOE1366 336178 1541 1554 CATAGCGCCTCTGA 3-8-3 MOE 1365 336177 1534 1547CCTCTGACTGGGAA 3-8-3 MOE 1364 389767 1534 1545 TCTGACTGGGAA 1-9-2 MOE1151 389968 1534 1545 TCTGACTGGGAA 1-10-1 MOE 1151 335344 1503 1516TCTCTGGTCCTTAC 2-10-2 MOE 1363 335355 1503 1516 TCTCTGGTCCTTAC 2-10-2MOE 1363 Phosphodiester linkage in wings 335370 1503 1516 TCTCTGGTCCTTAC2-10-2 Methyleneoxy BNA 1363 Phosphodiester linkage in wings 335381 15031516 TCTCTGGTCCTTAC 2-10-2 Methyleneoxy BNA 1363 335411 1503 1516TCTCTGGTCCTTAC 2-10-2 MOE 1363 3′ C is 9-(aminoethoxy)phenoxazine 3354121503 1516 TCTCTGGTCCTTAC 2-10-2 MOE 1363 C in 5′ wing is 9-(aminoethoxy)phenoxazine 335413 1503 1516 TCTCTGGTCCTTAC 2-10-2 MOE 1363C in wings are 9-(aminoethoxy)phenoxazine 336176 1502 1515CTCTGGTCCTTACT 3-8-3 MOE 1361 335345 1502 1517 GTCTCTGGTCCTTACT 3-10-3MOE 1362 335356 1502 1517 GTCTCTGGTCCTTACT 3-10-3 MOE 1362Phosphodiester linkage in wings 335371 1502 1517 GTCTCTGGTCCTTACT 3-10-3Methyleneoxy BNA 1362 Phosphodiester linkage in wings 335382 1502 1517GTCTCTGGTCCTTACT 3-10-3 Methyleneoxy BNA 1362 335414 1502 1517GTCTCTGGTCCTTACT 3-10-3 MOE 1362 C in 3′ wing is 9-(aminoethoxy)phenoxazine 335415 1502 1517 GTCTCTGGTCCTTACT 3-10-3 MOE1362 C in 5′ wing is 9- (aminoethoxy)phenoxazine 335416 1502 1517GTCTCTGGTCCTTACT 3-10-3 MOE 1362 C's in wings are9-(aminoethoxy)phenoxazine 336175 1495 1508 CCTTACTTCCCCAT 3-8-3 MOE1360 336174 1472 1485 GGGCCTCTTGTGCC 3-8-3 MOE 1359 336173 1465 1478TTGTGCCTTTAAAA 3-8-3 MOE 1358 398046 1465 1476 GTGCCTTTAAAA 1-10-1 MOE1199 389766 1464 1475 TGCCTTTAAAAA 1-9-2 MOE 1217 389967 1464 1475TGCCTTTAAAAA 1-10-1 MOE 1217 397976 1464 1477 TGTGCCTTTAAAAA 2-10-2 MOE1357 336172 1437 1450 AATAAATATGCACA 3-8-3 MOE 1356 398045 1423 1434TCATTACACCAG 1-10-1 MOE 1355 336171 1422 1435 ATCATTACACCAGT 3-8-3 MOE1354 389765 1422 1433 CATTACACCAGT 1-9-2 MOE 1353 389966 1422 1433CATTACACCAGT 1-10-1 MOE 1353 397975 1422 1435 ATCATTACACCAGT 2-10-2 MOE1354 390005 1400 1411 CCAGCTTTACAG 1-10-1 MOE 1352 336170 1392 1405TTACAGTGAATTGC 3-8-3 MOE 1351 398044 1382 1393 GCTGCAACATGA 1-10-1 MOE1350 336169 1381 1394 TGCTGCAACATGAT 3-8-3 MOE 1349 389764 1381 1392CTGCAACATGAT 1-9-2 MOE 1018 389965 1381 1392 CTGCAACATGAT 1-10-1 MOE1018 397974 1381 1394 TGCTGCAACATGAT 2-10-2 MOE 1349 336168 1362 1375TCTTCACTTAGCCA 3-8-3 MOE 1348 390004 1362 1373 TTCACTTAGCCA 1-10-1 MOE1208 336167 1353 1366 AGCCATTGGTCAAG 3-8-3 MOE 1347 398043 1345 1356CAAGATCTTCAC 1-10-1 MOE 1244 336166 1344 1357 TCAAGATCTTCACA 3-8-3 MOE1346 390003 1344 1355 AAGATCTTCACA 1-10-1 MOE 1243 397973 1344 1357TCAAGATCTTCACA 2-10-2 MOE 1346 336165 1329 1342 AAGGGTTTGATAAG 3-8-3 MOE1345 390002 1322 1333 ATAAGTTCTAGC 1-10-1 MOE 1344 336164 1318 1331AAGTTCTAGCTGTG 3-8-3 MOE 1343 398042 1305 1316 TGGGTTATGGTC 1-10-1 MOE1214 336163 1304 1317 GTGGGTTATGGTCT 3-8-3 MOE 1342 397972 1304 1317GTGGGTTATGGTCT 2-10-2 MOE 1342 398089 1298 1309 TGGTCTTCAAAA 1-10-1 MOE1341 389763 1296 1307 GTCTTCAAAAGG 1-9-2 MOE 1197 389964 1296 1307GTCTTCAAAAGG 1-10-1 MOE 1197 398041 1294 1305 CTTCAAAAGGAT 1-10-1 MOE1196 336162 1293 1306 TCTTCAAAAGGATA 3-8-3 MOE 1340 397971 1293 1306TCTTCAAAAGGATA 2-10-2 MOE 1340 398040 1279 1290 GTGCAACTCTGC 1-10-1 MOE1236 336161 1278 1291 TGTGCAACTCTGCA 3-8-3 MOE 1235 397970 1278 1291TGTGCAACTCTGCA 2-10-2 MOE 1235 398039 1264 1275 TAAATTTGGCGG 1-10-1 MOE1339 397969 1263 1276 TTAAATTTGGCGGT 2-10-2 MOE 1338 336160 1261 1274AAATTTGGCGGTGT 3-8-3 MOE 1337 336159 1253 1266 CGGTGTCATAATGT 3-8-3 MOE1336 398038 1252 1263 TGTCATAATGTC 1-10-1 MOE 1200 390000 1251 1262GTCATAATGTCT 1-10-1 MOE 1194 397968 1251 1264 GTGTCATAATGTCT 2-10-2 MOE1195 336158 1227 1240 AGATTGTATATCTT 3-8-3 MOE 1335 389762 1220 1231ATCTTGTAATGG 1-9-2 MOE 1334 389963 1220 1231 ATCTTGTAATGG 1-10-1 MOE1334 336157 1215 1228 TTGTAATGGTTTTT 3-8-3 MOE 1333 336156 1202 1215TATGCTTTGAATCC 3-8-3 MOE 1332 389998 1199 1210 TTTGAATCCAAA 1-10-1 MOE1331 397967 1198 1211 CTTTGAATCCAAAA 2-10-2 MOE 1330 336155 1190 1203CCAAAAACCTTACT 3-8-3 MOE 1500 336154 1176 1189 ACATCATCAATATT 3-8-3 MOE1329 389761 1171 1182 CAATATTGTTCC 1-9-2 MOE 1328 389962 1171 1182CAATATTGTTCC 1-10-1 MOE 1328 398037 1170 1181 AATATTGTTCCT 1-10-1 MOE1202 397966 1169 1182 CAATATTGTTCCTG 2-10-2 MOE 1327 336153 1164 1177TTGTTCCTGTATAC 3-8-3 MOE 1326 336152 1149 1162 CCTTCAAGTCTTTC 3-8-3 MOE1325 389996 1141 1152 TTTCTGCAGGAA 1-10-1 MOE 1165 336151 1138 1151TTCTGCAGGAAATC 3-8-3 MOE 1324 398036 1138 1149 CTGCAGGAAATC 1-10-1 MOE1323 397965 1137 1150 TCTGCAGGAAATCC 2-10-2 MOE 1322 389760 1129 1140ATCCCATAGCAA 1-9-2 MOE 1321 389961 1129 1140 ATCCCATAGCAA 1-10-1 MOE1321 398035 1126 1137 CCATAGCAATAA 1-10-1 MOE 1320 336150 1125 1138CCCATAGCAATAAT 3-8-3 MOE 1319 397964 1125 1138 CCCATAGCAATAAT 2-10-2 MOE1319 336149 1110 1123 TTTGGATAAATATA 3-8-3 MOE 1496 389995 1106 1117TAAATATAGGTC 1-10-1 MOE 1516 336148 1100 1113 TATAGGTCAAGTCT 3-8-3 MOE1495 398034 1099 1110 AGGTCAAGTCTA 1-10-1 MOE 1300 397963 1098 1111TAGGTCAAGTCTAA 2-10-2 MOE 1494 389994 1095 1106 CAAGTCTAAGTC 1-10-1 MOE1299 336147 1090 1103 GTCTAAGTCGAATC 3-8-3 MOE 1298 389993 1083 1094GAATCCATCCTC 1-10-1 MOE 1297 336146 1080 1093 AATCCATCCTCTTG 3-8-3 MOE1296 398033 1077 1088 ATCCTCTTGATA 1-10-1 MOE 1198 397962 1076 1089CATCCTCTTGATAT 2-10-2 MOE 1295 336145 1070 1083 CTTGATATCTCCTT 3-8-3 MOE1294 336144 1057 1070 TTTGTTTCTGCTAA 3-8-3 MOE 1293 389759 1056 1067GTTTCTGCTAAC 1-9-2 MOE 1292 389960 1056 1067 GTTTCTGCTAAC 1-10-1 MOE1292 392059 1055 1068 TGTTTCTGCTAACG 2-10-2 Methyleneoxy BNA 1291Unmodified cytosines in gap 336143 1044 1057 ACGATCTCTTTGAT 3-8-3 MOE1290 398032 1038 1049 TTTGATGATGGC 1-10-1 MOE 1222 397961 1037 1050CTTTGATGATGGCT 2-10-2 MOE 1289 389992 1036 1047 TGATGATGGCTG 1-10-1 MOE1288 336142 1032 1045 ATGATGGCTGTCAT 3-8-3 MOE 1287 389991 1021 1032TGTCTGGGAGCC 1-10-1 MOE 1286 392058 1020 1033 ATGTCTGGGAGCCT 2-10-2Methyleneoxy BNA 1285 Unmodified cytosines in gap 397960 1020 1033ATGTCTGGGAGCCT 2-10-2 MOE 1285 389990 1007 1018 TGGCTGAAGAAA 1-10-1 MOE1284 397959 1006 1019 GTGGCTGAAGAAAA 2-10-2 MOE 1283 398031 987 998GAGAGATGGCAG 1-10-1 MOE 1282 397958 986 999 AGAGAGATGGCAGA 2-10-2 MOE1281 389758 983 994 GATGGCAGAAGC 1-9-2 MOE 1280 389959 983 994GATGGCAGAAGC 1-10-1 MOE 1280 398030 976 987 GAAGCTGCTGGT 1-10-1 MOE 1143397957 975 988 AGAAGCTGCTGGTG 2-10-2 MOE 1279 389989 953 964TTCTGCAGGATG 1-10-1 MOE 1170 389757 941 952 GAAATGGCTCTG 1-9-2 MOE 1278389958 941 952 GAAATGGCTCTG 1-10-1 MOE 1278 397956 940 953GGAAATGGCTCTGG 2-10-2 MOE 1277 398029 931 942 TGGACTTGGCGG 1-10-1 MOE1186 397955 930 943 CTGGACTTGGCGGT 2-10-2 MOE 1276 398028 914 925GATGCCCCTCGC 1-10-1 MOE 1275 397954 913 926 TGATGCCCCTCGCT 2-10-2 MOE1274 398027 883 894 GGACCGCAGCCG 1-10-1 MOE 1155 397953 882 895TGGACCGCAGCCGG 2-10-2 MOE 1273 389756 874 885 CCGGGTAATGGC 1-9-2 MOE1272 389957 874 885 CCGGGTAATGGC 1-10-1 MOE 1272 398026 867 878ATGGCTGCTGCG 1-10-1 MOE 1160 397952 866 879 AATGGCTGCTGCGG 2-10-2 MOE1271 389987 848 859 CTGGATGGTTGC 1-10-1 MOE 1270 389755 806 817AGAGGCCTGGCA 1-9-2 MOE 1269 389956 806 817 AGAGGCCTGGCA 1-10-1 MOE 1269389985 584 595 ATGGTGACAGGC 1-10-1 MOE 1268 398025 581 592 GTGACAGGCGAC1-10-1 MOE 1267 397951 580 593 GGTGACAGGCGACT 2-10-2 MOE 1266 389754 312323 TGCTCACAGGCG 1-9-2 MOE 1158 389955 312 323 TGCTCACAGGCG 1-10-1 MOE1158 398024 231 242 CAGCGGCTCAAC 1-10-1 MOE 1265 397950 230 243ACAGCGGCTCAACT 2-10-2 MOE 1264 389982 205 216 CATGGCTGCAGC 1-10-1 MOE1161 392056 204 217 TCATGGCTGCAGCT 2-10-2 Methyleneoxy BNA 1263 394424204 217 TCATGGCTGCAGCT 2-10-2 MOE 1263 396007 204 217 TCATGGCTGCAGCT2-10-2 (R)-CMOE BNA 1263 Unmodified cytosines 396008 204 217TCATGGCTGCAGCT 2-10-2 (S)-CMOE BNA 1263 Unmodified cytosines 396009 204217 TCATGGCTGCAGCT 2-10-2 α-L-methyleneoxy BNA 1263 Unmodified cytosines396566 204 217 TCATGGCTGCAGCT 2-10-2 Oxyamino BNA 1263 Unmodifiedcytosines 396567 204 217 TCATGGCTGCAGCT 2-10-2 N-Methyl-Oxyamino BNA1263 Unmodified cytosines 396568 204 217 TCATGGCTGCAGCT 2-10-2(6R)-6-Methyl 1263 Methyleneoxy BNA Unmodified cytosines 397913 204 217TCATGGCTGCAGCT 2-10-2 OMe 1263 Unmodified cytosines in gap 401974 204217 TCATGGCTGCAGCT 2-10-2 OMe 1263 Unmodified cytosines 403737 204 217TCATGGCTGCAGCT 2-10-2 Methyleneoxy BNA 1263 5-thiazole nucleobases inwings 404121 204 217 TCATGGCTGCAGCT 2-10-2 Methyleneoxy BNA 12635-methylcytosine in gaps 3′ Terminal THF phosphorothioate 404228 204 217TCATGGCTGCAGCT 2-10-2 Methyleneoxy BNA 1263 5-methylcytosinse in gaps5′-terminal reverse abasic 396024 204 217 TCATGGCTGCAGCT 2-10-2(6′S)-6′-methyl- 1263 Methyleneoxy BNA Unmodified cytosines 396569 204217 TCATGGCTGCAGCT 2-10-2 (5′S)-5′-methyl- 1263 Methyleneoxy BNAUnmodified cytosines 396577 204 217 TCATGGCTGCAGCT 2-10-1-1 MethyleneoxyBNA/ 1263 Methyleneoxy BNA/2′- (butylacetamido)-palmitamide/ Unmodifiedcytosines in gap 396576 204 217 TCATGGCTGCAGCT 1-1-10-22′-(butylacetamido)- 1263 palmitamide/Methyleneoxy BNA/ Methyleneoxy BNAUnmodified cytosines in gap 398023 191 202 CCGAGAGGAGAG 1-10-1 MOE 1262397949 190 203 TCCGAGAGGAGAGA 2-10-2 MOE 1261 398022 126 137AAGAGTCCCGCC 1-10-1 MOE 1260 397948 125 138 AAAGAGTCCCGCCA 2-10-2 MOE1259

TABLE 22 Short Antisense Compounds targeted to SEQ ID NO: 15 5′ 3′ SEQISIS Target Target ID No. Site Site Sequence (5′-3′) Gapmer Motif NO397948 525 538 AAAGAGTCCCGCCA 2-10-2 MOE 1259 398022 526 537AAGAGTCCCGCC 1-10-1 MOE 1260 397949 590 603 TCCGAGAGGAGAGA 2-10-2 MOE1261 398023 591 602 CCGAGAGGAGAG 1-10-1 MOE 1262 394424 604 617TCATGGCTGCAGCT 2-10-2 MOE 1263 397913 604 617 TCATGGCTGCAGCT 2-10-2 OMe1263 Unmodified cytosines in gap 401974 604 617 TCATGGCTGCAGCT 2-10-2Ome 1263 Unmodified cytosines 403737 604 617 TCATGGCTGCAGCT 2-10-2Methyleneoxy BNA 1263 5-thiazole nucleobases in wings 392056 604 617TCATGGCTGCAGCT 2-10-2 Methyleneoxy BNA 1263 Unmodified cytosines in gap396576 604 617 TCATGGCTGCAGCT 1-1-10-2 2′- 1263 (butylacetamido)-palmitamide/Methyleneoxy BNA/Methyleneoxy BNA Unmodified cytosines ingap 396577 604 617 TCATGGCTGCAGCT 2-10-1-2 Methyleneoxy 1263BNA/Methyleneoxy BNA/ 2′-(butylacetamido)- palmitamide/ Unmodifiedcytosines in gap 404121 604 617 TCATGGCTGCAGCT 2-10-2 Methyleneoxy BNA1263 5-methylcytosine in gaps 3′ Terminal THF phosphorothioate 404228604 617 TCATGGCTGCAGCT 2-10-2 Methyleneoxy BNA 1263 5-methylcytosinse ingaps 5′-terminal reverse abasic 396007 604 617 TCATGGCTGCAGCT 2-10-2(R)-CMOE BNA 1263 Unmodified cytosines 396008 604 617 TCATGGCTGCAGCT2-10-2 (S)-CMOE BNA 1263 Unmodified cytosines 396009 604 617TCATGGCTGCAGCT 2-10-2 α-L-methyleneoxy 1263 BNA Unmodified cytosines396024 604 617 TCATGGCTGCAGCT 2-10-2 (6′S)-6′-methyl- 1263 MethyleneoxyBNA Unmodified cytosines 396566 604 617 TCATGGCTGCAGCT 2-10-2 OxyaminoBNA 1263 Unmodified cytosines 396567 604 617 TCATGGCTGCAGCT 2-10-2N-Methyl-Oxyamino 1263 BNA Unmodified cytosines 396568 604 617TCATGGCTGCAGCT 2-10-2 (6R)-6-Methyl 1263 Methyleneoxy BNA Unmodifiedcytosines 396569 604 617 TCATGGCTGCAGCT 2-10-2 (5′S)-5′-methyl- 1263Methyleneoxy BNA Unmodified cytosines 389982 605 616 CATGGCTGCAGC 1-10-1MOE 1161 397950 630 643 ACAGCGGCTCAACT 2-10-2 MOE 1264 398024 631 642CAGCGGCTCAAC 1-10-1 MOE 1265 389955 712 723 TGCTCACAGGCG 1-10-1 MOE 1158389754 712 723 TGCTCACAGGCG 1-9-2 MOE 1158 397951 980 993 GGTGACAGGCGACT2-10-2 MOE 1266 398025 981 992 GTGACAGGCGAC 1-10-1 MOE 1267 389985 984995 ATGGTGACAGGC 1-10-1 MOE 1268 389956 1206 1217 AGAGGCCTGGCA 1-10-1MOE 1269 389755 1206 1217 AGAGGCCTGGCA 1-9-2 MOE 1269 389987 1248 1259CTGGATGGTTGC 1-10-1 MOE 1270 397952 1266 1279 AATGGCTGCTGCGG 2-10-2 MOE1271 398026 1267 1278 ATGGCTGCTGCG 1-10-1 MOE 1160 389957 1274 1285CCGGGTAATGGC 1-10-1 MOE 1272 389756 1274 1285 CCGGGTAATGGC 1-9-2 MOE1272 397953 1282 1295 TGGACCGCAGCCGG 2-10-2 MOE 1273 398027 1283 1294GGACCGCAGCCG 1-10-1 MOE 1155 397954 1313 1326 TGATGCCCCTCGCT 2-10-2 MOE1274 398028 1314 1325 GATGCCCCTCGC 1-10-1 MOE 1275 397955 1330 1343CTGGACTTGGCGGT 2-10-2 MOE 1276 398029 1331 1342 TGGACTTGGCGG 1-10-1 MOE1186 397956 1340 1353 GGAAATGGCTCTGG 2-10-2 MOE 1277 389958 1341 1352GAAATGGCTCTG 1-10-1 MOE 1278 389757 1341 1352 GAAATGGCTCTG 1-9-2 MOE1278 389989 1353 1364 TTCTGCAGGATG 1-10-1 MOE 1170 397957 1375 1388AGAAGCTGCTGGTG 2-10-2 MOE 1279 398030 1376 1387 GAAGCTGCTGGT 1-10-1 MOE1143 389959 1383 1394 GATGGCAGAAGC 1-10-1 MOE 1280 389758 1383 1394GATGGCAGAAGC 1-9-2 MOE 1280 397958 1386 1399 AGAGAGATGGCAGA 2-10-2 MOE1281 398031 1387 1398 GAGAGATGGCAG 1-10-1 MOE 1282 397959 1406 1419GTGGCTGAAGAAAA 2-10-2 MOE 1283 389990 1407 1418 TGGCTGAAGAAA 1-10-1 MOE1284 397960 1420 1433 ATGTCTGGGAGCCT 2-10-2 MOE 1285 392058 1420 1433ATGTCTGGGAGCCT 2-10-2 Methyleneoxy BNA 1285 5-methylcytosine in wing389991 1421 1432 TGTCTGGGAGCC 1-10-1 MOE 1286 336142 1432 1445ATGATGGCTGTCAT 3-8-3 MOE 1287 389992 1436 1447 TGATGATGGCTG 1-10-1 MOE1288 397961 1437 1450 CTTTGATGATGGCT 2-10-2 MOE 1289 398032 1438 1449TTTGATGATGGC 1-10-1 MOE 1222 336143 1444 1457 ACGATCTCTTTGAT 3-8-3 MOE1290 392059 1455 1468 TGTTTCTGCTAACG 2-10-2 Methyleneoxy BNA 12915-methylcytosine in wing 389960 1456 1467 GTTTCTGCTAAC 1-10-1 MOE 1292389759 1456 1467 GTTTCTGCTAAC 1-9-2 MOE 1292 336144 1457 1470TTTGTTTCTGCTAA 3-8-3 MOE 1293 336145 1470 1483 CTTGATATCTCCTT 3-8-3 MOE1294 397962 1476 1489 CATCCTCTTGATAT 2-10-2 MOE 1295 398033 1477 1488ATCCTCTTGATA 1-10-1 MOE 1198 336146 1480 1493 AATCCATCCTCTTG 3-8-3 MOE1296 389993 1483 1494 GAATCCATCCTC 1-10-1 MOE 1297 336147 1490 1503GTCTAAGTCGAATC 3-8-3 MOE 1298 389994 1495 1506 CAAGTCTAAGTC 1-10-1 MOE1299 398034 1499 1510 AGGTCAAGTCTA 1-10-1 MOE 1300 398010 1500 1513TACAGGTCAAGTCT 2-10-2 MOE 1166 398077 1501 1512 ACAGGTCAAGTC 1-10-1 MOE1167 398011 1512 1525 CGCAGAAATGGATA 2-10-2 MOE 1301 398078 1513 1524GCAGAAATGGAT 1-10-1 MOE 1302 398012 1570 1583 TTCGCATCCGTCTA 2-10-2 MOE1303 398079 1571 1582 TCGCATCCGTCT 1-10-1 MOE 1304 398013 1663 1676CCCTAGGTTGAATA 2-10-2 MOE 1305 398080 1664 1675 CCTAGGTTGAAT 1-10-1 MOE1306 398014 2025 2038 GTTATGCAAATCAG 2-10-2 MOE 1307 398081 2026 2037TTATGCAAATCA 1-10-1 MOE 1308 398015 2620 2633 TGACTCAGTAAATT 2-10-2 MOE1309 398082 2621 2632 GACTCAGTAAAT 1-10-1 MOE 1310 398016 2655 2668TTAAAATTCTTGGG 2-10-2 MOE 1311 398083 2656 2667 TAAAATTCTTGG 1-10-1 MOE1312 398017 2687 2700 CCTAACTTTTAGAC 2-10-2 MOE 1313 398084 2688 2699CTAACTTTTAGA 1-10-1 MOE 1314 398018 2745 2758 ACCTGAAACTGCAA 2-10-2 MOE1315 398085 2746 2757 CCTGAAACTGCA 1-10-1 MOE 1157 398019 13166 13179GTGTCAAAACCACT 2-10-2 MOE 1316 398086 13167 13178 TGTCAAAACCAC 1-10-1MOE 1204 398020 14675 14688 CCTATTCCCACTGA 2-10-2 MOE 1317 398087 1467614687 CTATTCCCACTG 1-10-1 MOE 1318 390033 15351 15362 AGCCAACTGCAA1-10-1 MOE 1483 398021 30985 30998 TTGGATAAATATCT 2-10-2 MOE 1168 39808830986 30997 TGGATAAATATC 1-10-1 MOE 1169 397964 31001 31014CCCATAGCAATAAT 2-10-2 MOE 1319 336150 31001 31014 CCCATAGCAATAAT 3-8-3MOE 1319 398035 31002 31013 CCATAGCAATAA 1-10-1 MOE 1320 389961 3100531016 ATCCCATAGCAA 1-10-1 MOE 1321 389760 31005 31016 ATCCCATAGCAA 1-9-2MOE 1321 397965 31013 31026 TCTGCAGGAAATCC 2-10-2 MOE 1322 398036 3101431025 CTGCAGGAAATC 1-10-1 MOE 1323 336151 31014 31027 TTCTGCAGGAAATC3-8-3 MOE 1324 389996 31017 31028 TTTCTGCAGGAA 1-10-1 MOE 1165 33615231025 31038 CCTTCAAGTCTTTC 3-8-3 MOE 1325 336153 31040 31053TTGTTCCTGTATAC 3-8-3 MOE 1326 397966 31045 31058 CAATATTGTTCCTG 2-10-2MOE 1327 398037 31046 31057 AATATTGTTCCT 1-10-1 MOE 1202 389962 3104731058 CAATATTGTTCC 1-10-1 MOE 1328 389761 31047 31058 CAATATTGTTCC 1-9-2MOE 1328 336154 31052 31065 ACATCATCAATATT 3-8-3 MOE 1329 389977 3148031491 CTTAAAATTTGG 1-10-1 MOE 1421 389776 31480 31491 CTTAAAATTTGG 1-9-2MOE 1421 397967 62446 62459 CTTTGAATCCAAAA 2-10-2 MOE 1330 389998 6244762458 TTTGAATCCAAA 1-10-1 MOE 1331 336156 62450 62463 TATGCTTTGAATCC3-8-3 MOE 1332 336157 62463 62476 TTGTAATGGTTTTT 3-8-3 MOE 1333 38996362468 62479 ATCTTGTAATGG 1-10-1 MOE 1334 389762 62468 62479 ATCTTGTAATGG1-9-2 MOE 1334 336158 62475 62488 AGATTGTATATCTT 3-8-3 MOE 1335 39000067987 67998 GTCATAATGTCT 1-10-1 MOE 1194 397968 67987 68000GTGTCATAATGTCT 2-10-2 MOE 1195 398038 67988 67999 TGTCATAATGTC 1-10-1MOE 1200 336159 67989 68002 CGGTGTCATAATGT 3-8-3 MOE 1336 336160 6799768010 AAATTTGGCGGTGT 3-8-3 MOE 1337 397969 67999 68012 TTAAATTTGGCGGT2-10-2 MOE 1338 398039 68000 68011 TAAATTTGGCGG 1-10-1 MOE 1339 39797169952 69965 TCTTCAAAAGGATA 2-10-2 MOE 1340 336162 69952 69965TCTTCAAAAGGATA 3-8-3 MOE 1340 398041 69953 69964 CTTCAAAAGGAT 1-10-1 MOE1196 389964 69955 69966 GTCTTCAAAAGG 1-10-1 MOE 1197 389763 69955 69966GTCTTCAAAAGG 1-9-2 MOE 1197 398089 69957 69968 TGGTCTTCAAAA 1-10-1 MOE1341 397972 69963 69976 GTGGGTTATGGTCT 2-10-2 MOE 1342 336163 6996369976 GTGGGTTATGGTCT 3-8-3 MOE 1342 398042 69964 69975 TGGGTTATGGTC1-10-1 MOE 1214 336164 69977 69990 AAGTTCTAGCTGTG 3-8-3 MOE 1343 39000269981 69992 ATAAGTTCTAGC 1-10-1 MOE 1344 336165 69988 70001AAGGGTTTGATAAG 3-8-3 MOE 1345 390003 70003 70014 AAGATCTTCACA 1-10-1 MOE1243 397973 70003 70016 TCAAGATCTTCACA 2-10-2 MOE 1346 336166 7000370016 TCAAGATCTTCACA 3-8-3 MOE 1346 398043 70004 70015 CAAGATCTTCAC1-10-1 MOE 1244 336167 70012 70025 AGCCATTGGTCAAG 3-8-3 MOE 1347 39000470021 70032 TTCACTTAGCCA 1-10-1 MOE 1208 336168 70021 70034TCTTCACTTAGCCA 3-8-3 MOE 1348 389965 70040 70051 CTGCAACATGAT 1-10-1 MOE1018 389764 70040 70051 CTGCAACATGAT 1-9-2 MOE 1018 397974 70040 70053TGCTGCAACATGAT 2-10-2 MOE 1349 336169 70040 70053 TGCTGCAACATGAT 3-8-3MOE 1349 398044 70041 70052 GCTGCAACATGA 1-10-1 MOE 1350 336170 7005170064 TTACAGTGAATTGC 3-8-3 MOE 1351 390005 70059 70070 CCAGCTTTACAG1-10-1 MOE 1352 389966 70081 70092 CATTACACCAGT 1-10-1 MOE 1353 38976570081 70092 CATTACACCAGT 1-9-2 MOE 1353 397975 70081 70094ATCATTACACCAGT 2-10-2 MOE 1354 336171 70081 70094 ATCATTACACCAGT 3-8-3MOE 1354 398045 70082 70093 TCATTACACCAG 1-10-1 MOE 1355 336172 7009670109 AATAAATATGCACA 3-8-3 MOE 1356 389967 70123 70134 TGCCTTTAAAAA1-10-1 MOE 1217 389766 70123 70134 TGCCTTTAAAAA 1-9-2 MOE 1217 39797670123 70136 TGTGCCTTTAAAAA 2-10-2 MOE 1357 398046 70124 70135GTGCCTTTAAAA 1-10-1 MOE 1199 336173 70124 70137 TTGTGCCTTTAAAA 3-8-3 MOE1358 336174 70131 70144 GGGCCTCTTGTGCC 3-8-3 MOE 1359 336175 70154 70167CCTTACTTCCCCAT 3-8-3 MOE 1360 335345 70161 70176 GTCTCTGGTCCTTACT 3-10-3MOE 1362 335356 70161 70176 GTCTCTGGTCCTTACT 3-10-3 MOE 1362Phosphodiester linkage in wings 335414 70161 70176 GTCTCTGGTCCTTACT3-10-3 MOE 1362 C in 3′ wing is 9- (aminoethoxy)phenoxazine 335415 7016170176 GTCTCTGGTCCTTACT 3-10-3 MOE 1362 C in 5′ wing is 9-(aminoethoxy)phenoxazine 335416 70161 70176 GTCTCTGGTCCTTACT 3-10-3 MOE1362 C's in wings are 9- (aminoethoxy)phenoxazine 336176 70161 70174CTCTGGTCCTTACT 3-8-3 MOE 1361 335371 70161 70176 GTCTCTGGTCCTTACT 3-10-3Methyleneoxy BNA 1362 Phosphodiester linkage in wings 335382 70161 70176GTCTCTGGTCCTTACT 3-10-3 Methyleneoxy BNA 1362 335344 70162 70175TCTCTGGTCCTTAC 2-10-2 MOE 1363 335355 70162 70175 TCTCTGGTCCTTAC 2-10-2MOE 1363 Phosphodiester linkage in wings 335411 70162 70175TCTCTGGTCCTTAC 2-10-2 MOE 1363 3′ C is 9- (aminoethoxy)phenoxazine335412 70162 70175 TCTCTGGTCCTTAC 2-10-2 MOE 1363 2^(nd) C is 9-(aminoethoxy)phenoxazine 335413 70162 70175 TCTCTGGTCCTTAC 2-10-2 MOE1363 2^(nd) and 3′ terminal C's are 9- (aminoethoxy)phenoxazine 33537070162 70175 TCTCTGGTCCTTAC 2-10-2 Methyleneoxy BNA 1363 Phosphodiesterlinkage in wings 335381 70162 70175 TCTCTGGTCCTTAC 2-10-2 MethyleneoxyBNA 1363 398068 79799 79810 ACAGCTACACAA 1-10-1 MOE 1472 389968 8905689067 TCTGACTGGGAA 1-10-1 MOE 1151 389767 89056 89067 TCTGACTGGGAA 1-9-2MOE 1151 336177 89056 89069 CCTCTGACTGGGAA 3-8-3 MOE 1364 336178 8906389076 CATAGCGCCTCTGA 3-8-3 MOE 1365 336179 89083 89096 CAGGTAGCTATAAT3-8-3 MOE 1366 390007 89085 89096 CAGGTAGCTATA 1-10-1 MOE 1367 39000989135 89146 ATCTTGTGAAAC 1-10-1 MOE 1175 397977 89135 89148TCATCTTGTGAAAC 2-10-2 MOE 1368 336180 89135 89148 TCATCTTGTGAAAC 3-8-3MOE 1368 398047 89136 89147 CATCTTGTGAAA 1-10-1 MOE 1369 336181 8914589158 GTTTCAAACATCAT 3-8-3 MOE 1370 397978 89147 89160 TAGTTTCAAACATC2-10-2 MOE 1371 398048 89148 89159 AGTTTCAAACAT 1-10-1 MOE 1372 38996989152 89163 GAATAGTTTCAA 1-10-1 MOE 1373 389768 89152 89163 GAATAGTTTCAA1-9-2 MOE 1373 336182 89155 89168 CATTGGAATAGTTT 3-8-3 MOE 1374 39797989162 89175 CACTGAACATTGGA 2-10-2 MOE 1375 398049 89163 89174ACTGAACATTGG 1-10-1 MOE 1376 390010 89165 89176 CCACTGAACATT 1-10-1 MOE1240 336183 89166 89179 CCGCCACTGAACAT 3-8-3 MOE 1377 397980 94786 94799CAGACCACAAACTG 2-10-2 MOE 1378 398050 94787 94798 AGACCACAAACT 1-10-1MOE 1379 392060 94790 94803 CTGGCAGACCACAA 2-10-2 Methyleneoxy BNA 1380Unmodified cytosines in gap 389970 94791 94802 TGGCAGACCACA 1-10-1 MOE1249 389769 94791 94802 TGGCAGACCACA 1-9-2 MOE 1249 336185 94792 94805AGCTGGCAGACCAC 3-8-3 MOE 1381 397981 94798 94811 ACCTTTAGCTGGCA 2-10-2MOE 1382 398051 94799 94810 CCTTTAGCTGGC 1-10-1 MOE 1220 336186 9480394816 TCTTCACCTTTAGC 3-8-3 MOE 1383 390012 94860 94871 TCAAAGTACATG1-10-1 MOE 1384 336187 94862 94875 GAACTCAAAGTACA 3-8-3 MOE 1385 38997194865 94876 GGAACTCAAAGT 1-10-1 MOE 1386 389770 94865 94876 GGAACTCAAAGT1-9-2 MOE 1386 397982 94865 94878 AGGGAACTCAAAGT 2-10-2 MOE 1387 39805294866 94877 GGGAACTCAAAG 1-10-1 MOE 1388 336188 94869 94882GCTGAGGGAACTCA 3-8-3 MOE 1389 336189 94888 94901 TCACCACACACAGG 3-8-3MOE 1390 336190 94904 94917 GAACTCTACTTTGA 3-8-3 MOE 1391 389972 9490994920 GAAGAACTCTAC 1-10-1 MOE 1392 389771 94909 94920 GAAGAACTCTAC 1-9-2MOE 1392 397983 94910 94923 GTGGAAGAACTCTA 2-10-2 MOE 1393 398053 9491194922 TGGAAGAACTCT 1-10-1 MOE 1394 336191 94915 94928 TGTTTGTGGAAGAA3-8-3 MOE 1395 336192 94925 94938 CATCTTGTTCTGTT 3-8-3 MOE 1396 39798497824 97837 AGTGAAACATTTTG 2-10-2 MOE 1397 398054 97825 97836GTGAAACATTTT 1-10-1 MOE 1144 336194 97827 97840 AAAAGTGAAACATT 3-8-3 MOE1145 389973 97835 97846 TTACCCAAAAGT 1-10-1 MOE 1398 389772 97835 97846TTACCCAAAAGT 1-9-2 MOE 1398 336195 97836 97849 TATTTACCCAAAAG 3-8-3 MOE1399 397985 97837 97850 GTATTTACCCAAAA 2-10-2 MOE 1400 398055 9783897849 TATTTACCCAAA 1-10-1 MOE 1401 397986 97853 97866 TCCTGGTATGAAGA2-10-2 MOE 1402 336196 97853 97866 TCCTGGTATGAAGA 3-8-3 MOE 1402 39805697854 97865 CCTGGTATGAAG 1-10-1 MOE 1403 390015 97857 97868 GGTCCTGGTATG1-10-1 MOE 1404 336197 97862 97875 TTCCTCTGGTCCTG 3-8-3 MOE 1405 39798797866 97879 AGGTTTCCTCTGGT 2-10-2 MOE 1406 398057 97867 97878GGTTTCCTCTGG 1-10-1 MOE 1407 336198 97873 97886 TTTTCTGAGGTTTC 3-8-3 MOE1408 336199 97891 97904 AGACTTCCATTTTC 3-8-3 MOE 1409 389974 97893 97904AGACTTCCATTT 1-10-1 MOE 1410 389773 97893 97904 AGACTTCCATTT 1-9-2 MOE1410 336200 97918 97931 CAAATGCTATCGAT 3-8-3 MOE 1411 336201 97933 97946GCACGCTCTATACT 3-8-3 MOE 1412 389975 97934 97945 CACGCTCTATAC 1-10-1 MOE1413 389774 97934 97945 CACGCTCTATAC 1-9-2 MOE 1413 336202 97948 97961TCCTTGTCATTATC 3-8-3 MOE 1414 397988 97990 98003 GCTTTGTCAAGATC 2-10-2MOE 1415 389976 97991 98002 CTTTGTCAAGAT 1-10-1 MOE 1177 389775 9799198002 CTTTGTCAAGAT 1-9-2 MOE 1177 336203 97991 98004 TGCTTTGTCAAGAT3-8-3 MOE 1416 397989 98017 98030 AAGTATCGGTTGGC 2-10-2 MOE 1417 33620498017 98030 AAGTATCGGTTGGC 3-8-3 MOE 1417 398058 98018 98029AGTATCGGTTGG 1-10-1 MOE 1418 336205 98032 98045 TTAAAATTTGGAGA 3-8-3 MOE1419 397990 98034 98047 CCTTAAAATTTGGA 2-10-2 MOE 1420 389977 9803598046 CTTAAAATTTGG 1-10-1 MOE 1421 389776 98035 98046 CTTAAAATTTGG 1-9-2MOE 1421 336207 102230 102243 TCTACTGTTTTTGT 3-8-3 MOE 1422 336208102236 102249 GGCTCCTCTACTGT 3-8-3 MOE 1423 335330 102251 102265AGCCTCTGGATTTGA 1-10-4 MOE 1424 335331 102252 102266 TAGCCTCTGGATTTG1-10-4 MOE 1426 336209 102252 102265 AGCCTCTGGATTTG 3-8-3 MOE 1425335377 102252 102266 TAGCCTCTGGATTTG 1-10-4 Methyleneoxy BNA 1426Phosphodiester in 3′ wing 335376 102252 102266 TAGCCTCTGGATTTG 1-10-4Methyleneoxy BNA 1426 390577 102253 102266 TAGCCTCTGGATTT 1-10-3 MOE1427 Unmodified cytosines T's in wings are 2- thiothymines 335332 102253102267 CTAGCCTCTGGATTT 1-10-4 MOE 1429 386770 102253 102266TAGCCTCTGGATTT 1-11-2 MOE 1427 375560 102253 102267 CTAGCCTCTGGATTT2-10-3 MOE 1429 391449 102253 102267 CTAGCCTCTGGATTT 2-10-3 MOE 1429Unmodified cytosines 392055 102253 102267 CTAGCCTCTGGATTT 2-10-3 MOE1429 Unmodified cytosines in gap 362977 102253 102268 GCTAGCCTCTGGATTT2-12-2 MOE 1428 371975 102253 102267 CTAGCCTCTGGATTT 3-10-2 MOE 1429386556 102253 102268 GCTAGCCTCTGGATTT 3-10-3 MOE 1428 335341 102253102268 GCTAGCCTCTGGATTT 3-10-3 MOE 1428 335350 102253 102268GCTAGCCTCTGGATTT 3-10-3 MOE 1428 383739 102253 102268 GCTAGCCTCTGGATTT3-10-3 MOE 1428 5-methylcytosine in gap 390576 102253 102268GCTAGCCTCTGGATTT 3-10-3 MOE 1428 5-methylcytosine in gap T's in wingsare 2- thiothymines 390580 102253 102268 GCTAGCCTCTGGATTT 3-10-3 MOE1428 Pyrimidines in wings are 5- thiazole Unmodified cytosines in gap390581 102253 102268 GCTAGCCTCTGGATTT 3-10-3 MOE 1428 Unmodifiedcytosines in gap 391096 102253 102268 GCTAGCCTCTGGATTT 3-10-3 MOE 1428391098 102253 102268 GCTAGCCTCTGGATTT 3-10-3 MOE 1428 391863 102253102268 GCTAGCCTCTGGATTT 3-10-3 MOE 1428 Unmodified cytosines 384071102253 102268 GCTAGCCTCTGGATTT 3-10-3 OMe 1428 5-methylcytosine in gap385036 102253 102268 GCTAGCCTCTGGATTT 1-2-10-3 OMe/2′-O-methyl- 14284′-thio/2′-O-methyl-4′-thio Unmodified cytosines in wing 335368 102253102268 GCTAGCCTCTGGATTT 3-10-3 Methyleneoxy BNA 1428 Phosphodiesterlinkages in wings 391864 102253 102268 GCTAGCCTCTGGATTT 3-10-3Methyleneoxy BNA 1428 Unmodified cytosines in gap 392054 102253 102267CTAGCCTCTGGATTT 2-10-3 Methyleneoxy BNA 1429 Unmodified cytosines in gap391172 102253 102267 CTAGCCTCTGGATTT 2-10-3 Methyleneoxy BNA 1429Unmodified cytosines 391865 102253 102268 GCTAGCCTCTGGATTT 3-10-3Methyleneoxy BNA 1428 Unmodified cytosines 391868 102253 102268GCTAGCCTCTGGATTT 1-2-10-3 (5′R)-5′-methyl- 1428 Methyleneoxy BNA/Methyleneoxy BNA/(5′R)- 5′-methyl-Methyleneoxy BNA Unmodified cytosines391869 102253 102268 GCTAGCCTCTGGATTT 1-2-10-3 Methyleneoxy 1428BNA/(5′S)-5′-methyl- Methyleneoxy BNA/(5′S)- 5′-methyl-Methyleneoxy BNAUnmodified cytosines 384073 102253 102268 GCTAGCCTCTGGATTT 3-10-3Methyleneoxy BNA 1428 5-methylcytosine in gap 335379 102253 102268GCTAGCCTCTGGATTT 3-10-3 Methyleneoxy BNA 1428 390579 102253 102268GCTAGCCTCTGGATTT 1-1-1-10-3 MOE/4′thio/2′- 1428 O-[(2-methoxy)ethyl]-4′-thio/2′-O-[(2- methoxy)ethyl]-4′-thio Unmodified cytosines in wingsPhosphorodiester linkage in wings 390582 102253 102268 GCTAGCCTCTGGATTT1-2-10-3 MOE/4′thio/2′-O- 1428 [(2-methoxy)ethyl]-4′-thio Unmodifiedcytosines in wings Phosphorodiester linkage in wings 390606 102253102268 GCTAGCCTCTGGATTT 1-2-10-3 1428 MOE/pentaF/pentaF Unmodifiedcytosines in wings Phosphodiester linkage in wings 384072 102253 102268GCTAGCCTCTGGATTT 1-2-10-3 1428 MOE/pentaF/pentaF Unmodified cytosines inwings 385871 102253 102268 GCTAGCCTCTGGATTT 1-2-10-3 OMe/2′-O-[(2- 1428methoxy)ethyl]-4′-thio/2′-O- [(2-methoxy)ethyl]-4′-thio Unmodifiedcytosines in wing 390607 102253 102268 GCTAGCCTCTGGATTT 3-10-3MOE/pentaF 1428 Unmodified cytosines in wing 390608 102253 102268GCTAGCCTCTGGATTT 1-2-10-3 1428 MOE/pentaF/pentaF Unmodified cytosines inwing 390609 102253 102268 GCTAGCCTCTGGATTT 3-10-2-1 MOE/MOE/pentaF 1428Unmodified cytosines in wing 386682 102253 102268 GCTAGCCTCTGGATTT1-2-10-3 2′- 1428 (butylacetamido)- palmitamide/MOE/MOE 391173 102253102267 CTAGCCTCTGGATTT 2-10-3 (5′R)-5′-methyl- 1429 Methyleneoxy BNAUnmodified cytosines 391174 102253 102267 CTAGCCTCTGGATTT 2-10-3(5′S)-5′-methyl- 1429 Methyleneoxy BNA Unmodified cytosines 386970102254 102266 TAGCCTCTGGATT 1-10-2 MOE 1432 390578 102254 102266TAGCCTCTGGATT 1-10-2 MOE 1432 Unmodified cytosines Ts in wings are 2-thiothymines 335333 102254 102268 GCTAGCCTCTGGATT 1-10-4 MOE 1430 331429102254 102267 CTAGCCTCTGGATT 2-10-2 MOE 1431 335349 102254 102267CTAGCCTCTGGATT 2-10-2 MOE 1431 335367 102254 102267 CTAGCCTCTGGATT2-10-2 Methyleneoxy BNA 1431 Phosphodiester linkages in wings 392061102254 102267 CTAGCCTCTGGATT 2-10-2 Methyleneoxy BNA 1431 Unmodifiedcytosines in gap 335378 102254 102267 CTAGCCTCTGGATT 2-10-2 MethyleneoxyBNA 1431 383991 102254 102266 TAGCCTCTGGATT 1-10-2 14322′-(acetylamino-butyl- acetamido)-cholesterol/ MOE 383992 102254 102266TAGCCTCTGGATT 1-10-2 1432 2′-(acetylamino-butyl- acetamido)-cholicacid/MOE 386683 102254 102266 TAGCCTCTGGATT 1-10-2 1432 5′ terminal 2′-(butylacetamido)- palmitamide/MOE 390614 102254 102266 TAGCCTCTGGATT1-10-2 PentaF 1432 389954 102255 102266 TAGCCTCTGGAT 1-10-1 MOE 1434335334 102255 102269 TGCTAGCCTCTGGAT 1-10-4 MOE 1433 389777 102255102266 TAGCCTCTGGAT 1-9-2 MOE 1434 390430 102256 102268 GCTAGCCTCTGGA1-10-2 MOE 1163 Unmodified cytosines 390431 102256 102268 GCTAGCCTCTGGA1-10-2 MOE 1163 Unmodified cytosines C in wing 9-(aminoethoxy)phenoxazine 390432 102256 102268 GCTAGCCTCTGGA 1-10-2 MOE1163 390433 102256 102268 GCTAGCCTCTGGA 1-10-2 MOE 1163 Unmodifiedcytosines Nt 6 is 9- (aminoethoxy)phenoxazine 390434 102256 102268GCTAGCCTCTGGA 1-10-2 MOE 1163 Unmodified cytosines Nt 7 is 9-(aminoethoxy)phenoxazine 390435 102256 102268 GCTAGCCTCTGGA 1-10-2 MOE1163 Unmodified cytosines Nt 9 is 9- (aminoethoxy)phenoxazine 335335102256 102270 CTGCTAGCCTCTGGA 1-10-4 MOE 1435 335336 102257 102271ACTGCTAGCCTCTGG 1-10-4 MOE 1436 335337 102258 102272 AACTGCTAGCCTCTG1-10-4 MOE 1437 335338 102259 102273 GAACTGCTAGCCTCT 1-10-4 MOE 1438335339 102260 102274 TGAACTGCTAGCCTC 1-10-4 MOE 1439 335340 102261102275 TTGAACTGCTAGCCT 1-10-4 MOE 1440 336210 102261 102274TGAACTGCTAGCCT 3-8-3 MOE 1441 397991 102264 102277 AGTTGAACTGCTAG 2-10-2MOE 1442 398059 102265 102276 GTTGAACTGCTA 1-10-1 MOE 1443 390017 102268102279 GAAGTTGAACTG 1-10-1 MOE 1444 336211 102269 102282 ACAGAAGTTGAACT3-8-3 MOE 1445 397992 102293 102306 TCATTGTCACTAAC 2-10-2 MOE 1446336212 102293 102306 TCATTGTCACTAAC 3-8-3 MOE 1446 398060 102294 102305CATTGTCACTAA 1-10-1 MOE 1447 389978 102301 102312 TCAGGTTCATTG 1-10-1MOE 1448 389778 102301 102312 TCAGGTTCATTG 1-9-2 MOE 1448 336213 102303102316 ATGATCAGGTTCAT 3-8-3 MOE 1449 397993 102307 102320 TATAATGATCAGGT2-10-2 MOE 1450 398061 102308 102319 ATAATGATCAGG 1-10-1 MOE 1451 336214102314 102327 GAATATCTATAATG 3-8-3 MOE 1139 390019 102320 102331GTCAGAATATCT 1-10-1 MOE 1173 397994 102322 102335 TGGTGTCAGAATAT 2-10-2MOE 1452 398062 102323 102334 GGTGTCAGAATA 1-10-1 MOE 1255 336215 102326102339 TCAGTGGTGTCAGA 3-8-3 MOE 1453 336216 102339 102352 CTCTGGATCAGAGT3-8-3 MOE 1454 390020 102340 102351 TCTGGATCAGAG 1-10-1 MOE 1149 336217102349 102362 AAGGTTCATTCTCT 3-8-3 MOE 1455 397995 102357 102370TTCATCAAAAGGTT 2-10-2 MOE 1456 389979 102358 102369 TCATCAAAAGGT 1-10-1MOE 1176 389779 102358 102369 TCATCAAAAGGT 1-9-2 MOE 1176 336218 102358102371 CTTCATCAAAAGGT 3-8-3 MOE 1457 390021 102360 102371 CTTCATCAAAAG1-10-1 MOE 1458 336219 102366 102379 ATGCTGATCTTCAT 3-8-3 MOE 1459336220 102381 102394 TTTTGTAATTTGTG 3-8-3 MOE 1460 336221 102387 102400TCAGACTTTTGTAA 3-8-3 MOE 1461 390022 102443 102454 CAGTTTATTCAA 1-10-1MOE 1142 397996 102477 102490 TGTCCTATTGCCAT 2-10-2 MOE 1462 398063102478 102489 GTCCTATTGCCA 1-10-1 MOE 1205 397997 102487 102500TCTGACACAATGTC 2-10-2 MOE 1463 398064 102488 102499 CTGACACAATGT 1-10-1MOE 1464 397998 102505 102518 TGTTCCTATAACTG 2-10-2 MOE 1465 398065102506 102517 GTTCCTATAACT 1-10-1 MOE 1466 397999 102528 102541AAGATTGGTCAGGA 2-10-2 MOE 1467 398066 102529 102540 AGATTGGTCAGG 1-10-1MOE 1468 398000 102561 102574 GTGTCAAAACCCTG 2-10-2 MOE 1469 398067102562 102573 TGTCAAAACCCT 1-10-1 MOE 1210 390025 102563 102574GTGTCAAAACCC 1-10-1 MOE 1211 390026 102595 102606 AGCTACACAACC 1-10-1MOE 1470 398001 102596 102609 CACAGCTACACAAC 2-10-2 MOE 1471 398068102597 102608 ACAGCTACACAA 1-10-1 MOE 1472 398002 102607 102620TATATACATGACAC 2-10-2 MOE 1473 398069 102608 102619 ATATACATGACA 1-10-1MOE 1474 390027 102612 102623 AGGTATATACAT 1-10-1 MOE 1206 398003 102637102650 AATTTTAAATGTCC 2-10-2 MOE 1475 398070 102638 102649 ATTTTAAATGTC1-10-1 MOE 1476 390028 102648 102659 TCCTAATTGAAT 1-10-1 MOE 1477 390029102667 102678 AAAGTGCCATCT 1-10-1 MOE 1478 398004 102689 102702TTTATAAAACTGGA 2-10-2 MOE 1479 398071 102690 102701 TTATAAAACTGG 1-10-1MOE 1480 390030 102691 102702 TTTATAAAACTG 1-10-1 MOE 1074 398005 102827102840 TGCAAACTTATCTG 2-10-2 MOE 1481 398072 102828 102839 GCAAACTTATCT1-10-1 MOE 1482 390033 102836 102847 AGCCAACTGCAA 1-10-1 MOE 1483 398006102837 102850 CTTAGCCAACTGCA 2-10-2 MOE 1484 398073 102838 102849TTAGCCAACTGC 1-10-1 MOE 1485 398007 103069 103082 AGCACCAATATGCT 2-10-2MOE 1247 398074 103070 103081 GCACCAATATGC 1-10-1 MOE 1248 398008 103267103280 TAAATCATTGTCAA 2-10-2 MOE 1486 398075 103268 103279 AAATCATTGTCA1-10-1 MOE 1233 398009 103327 103340 GCACTGGCCTTGAT 2-10-2 MOE 1487398076 103328 103339 CACTGGCCTTGA 1-10-1 MOE 1488 390041 103332 103343TTAGCACTGGCC 1-10-1 MOE 1489 390047 103585 103596 TGTGTAAGGTCA 1-10-1MOE 1490 390049 103636 103647 GTTAATGACATT 1-10-1 MOE 1491 390050 103660103671 GTATTCAAGTAA 1-10-1 MOE 1140 390052 103780 103791 GACAATTTCTAC1-10-1 MOE 1492 390054 103862 103873 AACACTGCACAT 1-10-1 MOE 1493

Salts, Prodrugs and Bioequivalents

The antisense compounds provided herein comprise any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any otherfunctional chemical equivalent which, upon administration to an animalincluding a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to prodrugs and pharmaceuticallyacceptable salts of the antisense compounds, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive or less active form that is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes, chemicals, and/or conditions. In particular, prodrug versionsof the oligonucleotides are prepared as SATE ((S-acetyl-2-thioethyl)phosphate) derivatives according to the methods disclosed in WO 93/24510or WO 94/26764. Prodrugs can also include antisense compounds whereinone or both ends comprise nucleobases that are cleaved (e.g., byincorporating phosphodiester backbone linkages at the ends) to producethe active compound. In certain embodiments, one or more non-drugmoieties is cleaved from a prodrug to yield the active form. In certainsuch embodiments, such non-drug moieties is not a nucleotide oroligonucleotide.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds described herein:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.Sodium salts of antisense oligonucleotides are useful and are wellaccepted for therapeutic administration to humans.

In certain embodiments, salts, including, but not limited to sodiumsalts, of double stranded nucleic acids (including but not limited todsRNA compounds) are also provided.

G. CERTAIN PHARMACEUTICAL COMPOSITIONS

In certain embodiments, pharmaceutical compositions of the presentinvention comprise one or more short antisense compound and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition of the presentinvention is prepared using known techniques, including, but not limitedto mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or tabletting processes.

In certain embodiments, a pharmaceutical composition of the presentinvention is a liquid (e.g., a suspension, elixir and/or solution). Incertain of such embodiments, a liquid pharmaceutical composition isprepared using ingredients known in the art, including, but not limitedto, water, glycols, oils, alcohols, flavoring agents, preservatives, andcoloring agents.

In certain embodiments, a pharmaceutical composition of the presentinvention is a solid (e.g., a powder, tablet, and/or capsule). Incertain of such embodiments, a solid pharmaceutical compositioncomprising one or more oligonucleotides is prepared using ingredientsknown in the art, including, but not limited to, starches, sugars,diluents, granulating agents, lubricants, binders, and disintegratingagents.

In certain embodiments, a pharmaceutical composition of the presentinvention is formulated as a depot preparation. Certain such depotpreparations are typically longer acting than non-depot preparations. Incertain embodiments, such preparations are administered by implantation(for example subcutaneously or intramuscularly) or by intramuscularinjection. In certain embodiments, depot preparations are prepared usingsuitable polymeric or hydrophobic materials (for example an emulsion inan acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a delivery system. Examples of delivery systemsinclude, but are not limited to, liposomes and emulsions. Certaindelivery systems are useful for preparing certain pharmaceuticalcompositions including those comprising hydrophobic compounds. Incertain embodiments, certain organic solvents such as dimethylsulfoxideare used.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises one or more tissue-specific delivery moleculesdesigned to deliver the one or more pharmaceutical agents of the presentinvention to specific tissues or cell types. For example, in certainembodiments, pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a co-solvent system. Certain of such co-solventsystems comprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™, and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition of the presentinvention comprises a sustained-release system. A non-limiting exampleof such a sustained-release system is a semi-permeable matrix of solidhydrophobic polymers. In certain embodiments, sustained-release systemsmay, depending on their chemical nature, release pharmaceutical agentsover a period of hours, days, weeks or months.

In certain embodiments, a pharmaceutical composition of the presentinvention is prepared for oral administration. In certain of suchembodiments, a pharmaceutical composition is formulated by combining oneor more oligonucleotides with one or more pharmaceutically acceptablecarriers. Certain of such carriers enable pharmaceutical compositions tobe formulated as tablets, pills, dragees, capsules, liquids, gels,syrups, slurries, suspensions and the like, for oral ingestion by asubject. In certain embodiments, pharmaceutical compositions for oraluse are obtained by mixing oligonucleotide and one or more solidexcipient. Suitable excipients include, but are not limited to, fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;cellulose preparations such as, for example, maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). In certain embodiments, such a mixture isoptionally ground and auxiliaries are optionally added. In certainembodiments, pharmaceutical compositions are formed to obtain tablets ordragee cores. In certain embodiments, disintegrating agents (e.g.,cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate) are added.

In certain embodiments, dragee cores are provided with coatings. Incertain such embodiments, concentrated sugar solutions may be used,which may optionally comprise gum arabic, talc, polyvinyl pyrrolidone,carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquersolutions, and suitable organic solvents or solvent mixtures. Dyestuffsor pigments may be added to tablets or dragee coatings.

In certain embodiments, pharmaceutical compositions for oraladministration are push-fit capsules made of gelatin. Certain of suchpush-fit capsules comprise one or more pharmaceutical agents of thepresent invention in admixture with one or more filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In certain embodiments,pharmaceutical compositions for oral administration are soft, sealedcapsules made of gelatin and a plasticizer, such as glycerol orsorbitol. In certain soft capsules, one or more pharmaceutical agents ofthe present invention are be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added.

In certain embodiments, pharmaceutical compositions are prepared forbuccal administration. Certain of such pharmaceutical compositions aretablets or lozenges formulated in conventional manner.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay comprise formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may comprise substances that increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, such suspensions may also comprisesuitable stabilizers or agents that increase the solubility of thepharmaceutical agents to allow for the preparation of highlyconcentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition is prepared foradministration by inhalation. Certain of such pharmaceuticalcompositions for inhalation are prepared in the form of an aerosol sprayin a pressurized pack or a nebulizer. Certain of such pharmaceuticalcompositions comprise a propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In certain embodiments using a pressurized aerosol,the dosage unit may be determined with a valve that delivers a meteredamount. In certain embodiments, capsules and cartridges for use in aninhaler or insufflator may be formulated. Certain of such formulationscomprise a powder mixture of a pharmaceutical agent of the invention anda suitable powder base such as lactose or starch.

In certain embodiments, a pharmaceutical composition is prepared forrectal administration, such as a suppositories or retention enema.Certain of such pharmaceutical compositions comprise known ingredients,such as cocoa butter and/or other glycerides.

In certain embodiments, a pharmaceutical composition is prepared fortopical administration. Certain of such pharmaceutical compositionscomprise bland moisturizing bases, such as ointments or creams.Exemplary suitable ointment bases include, but are not limited to,petrolatum, petrolatum plus volatile silicones, lanolin and water in oilemulsions such as Eucerin™, available from Beiersdorf (Cincinnati,Ohio). Exemplary suitable cream bases include, but are not limited to,Nivea™ Cream, available from Beiersdorf (Cincinnati, Ohio), cold cream(USP), Purpose Cream™, available from Johnson & Johnson (New Brunswick,N.J.), hydrophilic ointment (USP) and Lubriderm™, available from Pfizer(Morris Plains, N.J.).

In certain embodiments, a pharmaceutical composition of the presentinvention comprises an oligonucleotide in a therapeutically effectiveamount. In certain embodiments, the therapeutically effective amount issufficient to prevent, alleviate or ameliorate symptoms of a disease orto prolong the survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, one or more short antisense compound of thepresent invention is formulated as a prodrug. In certain embodiments,upon in vivo administration, a prodrug is chemically converted to thebiologically, pharmaceutically or therapeutically more active form ofthe short antisense compound. In certain embodiments, prodrugs areuseful because they are easier to administer than the correspondingactive form. For example, in certain instances, a prodrug may be morebioavailable (e.g., through oral administration) than is thecorresponding active form. In certain instances, a prodrug may haveimproved solubility compared to the corresponding active form. Incertain embodiments, prodrugs are less water soluble than thecorresponding active form. In certain instances, such prodrugs possesssuperior transmittal across cell membranes, where water solubility isdetrimental to mobility. In certain embodiments, a prodrug is an ester.In certain such embodiments, the ester is metabolically hydrolyzed tocarboxylic acid upon administration. In certain instances the carboxylicacid containing compound is the corresponding active form. In certainembodiments, a prodrug comprises a short peptide (polyaminoacid) boundto an acid group. In certain of such embodiments, the peptide is cleavedupon administration to form the corresponding active form.

In certain embodiments, a prodrug is produced by modifying apharmaceutically active compound such that the active compound will beregenerated upon in vivo administration. The prodrug can be designed toalter the metabolic stability or the transport characteristics of adrug, to mask side effects or toxicity, to improve the flavor of a drugor to alter other characteristics or properties of a drug. By virtue ofknowledge of pharmacodynamic processes and drug metabolism in vivo,those of skill in this art, once a pharmaceutically active compound isknown, can design prodrugs of the compound (see, e.g., Nogrady (1985)Medicinal Chemistry A Biochemical Approach, Oxford University Press, NewYork, pages 388-392).

In certain embodiments, a pharmaceutical composition comprising one ormore pharmaceutical agents of the present invention is useful fortreating a conditions or disorders in a mammalian, and particularly in ahuman, subject. Suitable administration routes include, but are notlimited to, oral, rectal, transmucosal, intestinal, enteral, topical,suppository, through inhalation, intrathecal, intraventricular,intraperitoneal, intranasal, intraocular and parenteral (e.g.,intravenous, intramuscular, intramedullary, and subcutaneous). Incertain embodiments, pharmaceutical intrathecals are administered toachieve local rather than systemic exposures. For example,pharmaceutical compositions may be injected directly in the area ofdesired effect (e.g., in the renal or cardiac area).

In certain embodiments, short antisense compounds, compared to theirparent oligonucleotides, make them particularly suited to oraladministration. In certain embodiments, short antisense compounds arebetter suited for oral administration than their parent oligonucleotidesbecause they have increased potency compared to those parentoligonucleotides. In certain embodiments, short antisense compounds arebetter suited for oral administration than their parent oligonucleotidesbecause they have better stability, availability or solubilityproperties compared to those parent oligonucleotides.

In a further aspect, a pharmaceutical agent is sterile lyophilizedoligonucleotide that is reconstituted with a suitable diluent, e.g.,sterile water for injection. The reconstituted product is administeredas a subcutaneous injection or as an intravenous infusion after dilutioninto saline. The lyophilized drug product consists of theoligonucleotide which has been prepared in water for injection, adjustedto pH 7.0-9.0 with acid or base during preparation, and thenlyophilized. The lyophilized oligonucleotide may be 25-800 mg of theoligonucleotide. It is understood that this encompasses 25, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mgof lyophilized oligonucleotide. The lyophilized drug product may bepackaged in a 2 mL Type I, clear glass vial (ammonium sulfate-treated),stoppered with a bromobutyl rubber closure and sealed with an aluminumFLIP-OFF® overseal.

The compositions of the present invention may additionally compriseother adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may comprise additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycomprise additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the oligonucleotide(s) of the formulation.

The antisense compounds provided herein may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds.

Also described herein are pharmaceutical compositions and formulationswhich include the antisense compounds provided herein. Thepharmaceutical compositions may be administered in a number of waysdepending upon whether local or systemic treatment is desired and uponthe area to be treated. In a preferred embodiment, administration istopical to the surface of the respiratory tract, particularly pulmonary,e.g., by nebulization, inhalation, or insufflation of powders oraerosols, by mouth and/or nose.

The pharmaceutical formulations described herein, which may convenientlybe presented in unit dosage form, may be prepared according toconventional techniques well known in the pharmaceutical industry. Suchtechniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers,finely divided solid carriers, or both, and then, if necessary, shapingthe product (e.g., into a specific particle size for delivery). In apreferred embodiment, the pharmaceutical formulations are prepared forpulmonary administration in an appropriate solvent, e.g., water ornormal saline, possibly in a sterile formulation, with carriers or otheragents to allow for the formation of droplets of the desired diameterfor delivery using inhalers, nasal delivery devices, nebulizers, andother devices for pulmonary delivery. Alternatively, the pharmaceuticalformulations may be formulated as dry powders for use in dry powderinhalers.

A “pharmaceutical carrier” or “excipient” can be a pharmaceuticallyacceptable solvent, suspending agent or any other pharmacologicallyinert vehicle for delivering one or more nucleic acids to an individualand are known in the art. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition.

H. CERTAIN THERAPEUTIC USES

In certain embodiments, antisense compounds are used to modulate theexpression of a target gene in an animal, such as a human. In certainembodiments, such compounds can be used to treat metabolic disorders ormodulate one or more disease indications. For example, the methodscomprise the step of administering to said animal in need of therapy fora disease or condition associated with a target gene an effective amountof an antisense compound that modulates expression of the target gene.Antisense compounds provided herein which effectively modulateexpression of a target RNA or protein products of expression areconsidered active antisense compounds. Active antisense compounds alsoinclude compounds which effectively modulate one or more of a number ofdisease indications, including metabolic and cardiovascular diseaseindications, examples of which are described below.

Modulation of expression of a target gene can be measured in a bodilyfluid, which may or may not contain cells; tissue; or organ of theanimal. Methods of obtaining samples for analysis, such as body fluids(e.g., sputum, serum, urine), tissues (e.g., biopsy), or organs, andmethods of preparation of the samples to allow for analysis are wellknown to those skilled in the art. Methods for analysis of RNA andprotein levels are discussed above and are well known to those skilledin the art. The effects of treatment can be assessed by measuringbiomarkers, or disease indications, associated with the target geneexpression in the aforementioned fluids, tissues or organs, collectedfrom an animal contacted with one or more compounds described herein, byroutine clinical methods known in the art. These biomarkers include butare not limited to: liver transaminases, bilirubin, albumin, blood ureanitrogen, creatine and other markers of kidney and liver function;interleukins, tumor necrosis factors, intracellular adhesion molecules,C-reactive protein, chemokines, cytokines, and other markers ofinflammation. The antisense compounds provided herein can be utilized inpharmaceutical compositions by adding an effective amount of a compoundto a suitable pharmaceutically acceptable diluent or carrier. Acceptablecarriers and diluents are well known to those skilled in the art.Selection of a diluent or carrier is based on a number of factors,including, but not limited to, the solubility of the compound and theroute of administration. Such considerations are well understood bythose skilled in the art. In one aspect, the antisense compoundsdescribed herein inhibit expression of a target gene. The compounds canalso be used in the manufacture of a medicament for the treatment ofdiseases and disorders related to a target gene.

Methods whereby bodily fluids, organs or tissues are contacted with aneffective amount of one or more of the antisense compounds orcompositions provided herein are also contemplated. Bodily fluids,organs or tissues can be contacted with one or more of the compoundsresulting in modulation of target gene expression in the cells of bodilyfluids, organs or tissues. An effective amount can be determined bymonitoring the modulatory effect of the antisense compound or compoundsor compositions on target nucleic acids or their products by methodsroutine to the skilled artisan.

Co-Administration

In certain embodiments, two or more antisense compounds areco-administered. In certain embodiments, pharmaceutical compositionsinclude one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more antisense compoundstargeted to a second nucleic acid target. One or more of those antisensecompounds may be a short antisense compound. In certain embodiments,pharmaceutical compositions include two or more antisense compoundstargeted to different regions of the same nucleic acid target. One ormore of such antisense compounds may be a short antisense compound. Twoor more combined compounds may be used together or sequentially.

In certain embodiments, one or more pharmaceutical compositions areco-administered with one or more other pharmaceutical agents. In certainembodiments, such one or more other pharmaceutical agents are designedto treat the same disease or condition as the one or more pharmaceuticalcompositions of the present invention. In certain embodiments, such oneor more other pharmaceutical agents are designed to treat a differentdisease or condition as the one or more pharmaceutical compositions ofthe present invention. In certain embodiments, such one or more otherpharmaceutical agents are designed to treat an undesired effect of oneor more pharmaceutical compositions of the present invention. In certainembodiments, one or more pharmaceutical compositions of the presentinvention are co-administered with another pharmaceutical agent to treatan undesired effect of that other pharmaceutical agent. In certainembodiments, one or more pharmaceutical compositions of the presentinvention and one or more other pharmaceutical agents are administeredat the same time. In certain embodiments, one or more pharmaceuticalcompositions of the present invention and one or more otherpharmaceutical agents are administered at different times. In certainembodiments, one or more pharmaceutical compositions of the presentinvention and one or more other pharmaceutical agents are preparedtogether in a single formulation. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are prepared separately.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition of the presentinvention include lipid-lowering agents. In certain such embodiments,pharmaceutical agents that may be co-administered with a pharmaceuticalcomposition of the present invention include, but are not limited toatorvastatin, simvastatin, rosuvastatin, and ezetimibe. In certain suchembodiments, the lipid-lowering agent is administered prior toadministration of a pharmaceutical composition of the present invention.In certain such embodiments, the lipid-lowering agent is administeredfollowing administration of a pharmaceutical composition of the presentinvention. In certain such embodiments the lipid-lowering agent isadministered at the same time as a pharmaceutical composition of thepresent invention. In certain such embodiments the dose of aco-administered lipid-lowering agent is the same as the dose that wouldbe administered if the lipid-lowering agent was administered alone. Incertain such embodiments the dose of a co-administered lipid-loweringagent is lower than the dose that would be administered if thelipid-lowering agent was administered alone. In certain such embodimentsthe dose of a co-administered lipid-lowering agent is greater than thedose that would be administered if the lipid-lowering agent wasadministered alone.

In certain embodiments, a co-administered lipid-lowering agent is aHMG-CoA reductase inhibitor. In certain such embodiments the HMG-CoAreductase inhibitor is a statin. In certain such embodiments the statinis selected from atorvastatin, simvastatin, pravastatin, fluvastatin,and rosuvastatin. In certain embodiments, a co-administeredlipid-lowering agent is a cholesterol absorption inhibitor. In certainsuch embodiments, cholesterol absorption inhibitor is ezetimibe. Incertain embodiments, a co-administered lipid-lowering agent is aco-formulated HMG-CoA reductase inhibitor and cholesterol absorptioninhibitor. In certain such embodiments the co-formulated lipid-loweringagent is ezetimibe/simvastatin. In certain embodiments, aco-administered lipid-lowering agent is a microsomal triglyceridetransfer protein inhibitor.

In certain embodiments, a co-administered pharmaceutical agent is a bileacid sequestrant. In certain such embodiments, the bile acid sequestrantis selected from cholestyramine, colestipol, and colesevelam.

In certain embodiments, a co-administered pharmaceutical agent is anicotinic acid. In certain such embodiments, the nicotinic acid isselected from immediate release nicotinic acid, extended releasenicotinic acid, and sustained release nicotinic acid.

In certain embodiments, a co-administered pharmaceutical agent is afibric acid. In certain such embodiments, a fibric acid is selected fromgemfibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.

Further examples of pharmaceutical agents that may be co-administeredwith a pharmaceutical composition of the present invention include, butare not limited to, corticosteroids, including but not limited toprednisone; immunoglobulins, including, but not limited to intravenousimmunoglobulin (IVIg); analgesics (e.g., acetaminophen);anti-inflammatory agents, including, but not limited to non-steroidalanti-inflammatory drugs (e.g., ibuprofen, COX-1 inhibitors, and COX-2,inhibitors); salicylates; antibiotics; antivirals; antifungal agents;antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins,sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics;hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin,progestin, somatostan, and thyroid hormones); immunomodulators; musclerelaxants; antihistamines; osteoporosis agents (e.g., biphosphonates,calcitonin, and estrogens); prostaglandins, antineoplastic agents;psychotherapeutic agents; sedatives; poison oak or poison sumacproducts; antibodies; and vaccines.

In certain embodiments, the pharmaceutical compositions of the presentinvention may be administered in conduction with a lipid-loweringtherapy. In certain such embodiments, a lipid-lowering therapy istherapeutic lifestyle change. In certain such embodiments, alipid-lowering therapy is LDL apheresis.

I. KITS, RESEARCH REAGENTS AND DIAGNOSTICS

The antisense compounds provided herein can be utilized for diagnostics,and as research reagents and kits. Furthermore, antisense compounds,which are able to inhibit gene expression or modulate gene expressionwith specificity, are often used by those of ordinary skill to elucidatethe function of particular genes or to distinguish between functions ofvarious members of a biological pathway.

For use in kits and diagnostics, the antisense compounds describedherein, either alone or in combination with other compounds ortherapeutics, can be used as tools in differential and/or combinatorialanalyses to elucidate expression patterns of a portion or the entirecomplement of genes expressed within cells and tissues. Methods of geneexpression analysis are well known to those skilled in the art.

J. CERTAIN ADVANTAGES OF SHORT ANTISENSE COMPOUNDS

In certain embodiments, short antisense compounds have advantages whencompared to their parent oligonucleotides. For example, in certainembodiments, short antisense compounds have greater affinity for atarget nucleic acid than their parent oligonucleotide. In certainembodiments, short antisense compounds have greater potency in vitrothan their parent oligonucleotide. In certain such embodiments, thatincreased in vitro potency is not entirely explained by increasedaffinity. In certain embodiments, such increased in vitro potency may beattributable to increased ability of short antisense compounds topenetrate cells and/or increased ability to access target nucleic acidsin a cell. In certain embodiments, short antisense compounds havegreater potency in vivo than their parent oligonucleotides. In certainembodiments, such greater in vivo potency is not attributable toincreased in vitro potency or increased affinity. In certainembodiments, short antisense compounds have even greater in vivo potencycompared to their parent oligonucleotides than would be predicted basedon in vitro potencies or on affinities. In certain embodiments, suchincreased in vivo potency may be attributable to increasedbioavailability, better penetration into the cell, better access totarget nucleic acid once in the cell, or other factors.

In certain embodiments, one would expect short antisense compounds to beless specific for their target nucleic acid compared to their parentoligonucleotides. In certain such embodiments, one would expectincreased side-effects, including potential for toxic effects, fromshort antisense compounds. In certain embodiments, such additionalside-effects are not observed. In certain embodiments, non-targetnucleic acids to which a particular short antisense compound may bindare not available to the short antisense compound. In such embodiments,side-effects, including toxicity, are less problematic than would bepredicted.

In certain embodiments, because they are smaller, short antisensecompounds are less likely to bind proteins. In certain such embodiments,such less binding of proteins results in lower toxicity, since proteinbinding may have undesired consequences. In certain embodiments, suchless binding of proteins results in greater potency, since it leavesmore antisense compound available for therapeutic effect. In certainembodiments, less binding of proteins results in decreased drug-druginteraction toxicity.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

Example 1 Cell Culture and Treatment with Short Antisense Compounds

The effect of short antisense compounds on target nucleic acidexpression can be tested in any one of a number of cultured or primarycell lines. Cells lines can be obtained from publicly available sources,such as the American Type Culture Collection (Manassas, Va.). Cells arecultured according to methods well known to those of ordinary skill inthe art.

When cells reached appropriate confluency, they were treated witholigonucleotide using LIPOFECTIN® as described. When cells reached65-75% confluency, they were treated with oligonucleotide.Oligonucleotide was mixed with LIPOFECTIN® Invitrogen Life Technologies,Carlsbad, Calif.) in Opti-MEM®-1 reduced serum medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) to achieve the desired concentration ofoligonucleotide and a LIPOFECTIN® concentration of 2.5 or 3 μg/mL per100 nM oligonucleotide. This transfection mixture was incubated at roomtemperature for approximately 0.5 hours. For cells grown in 96-wellplates, wells were washed once with 100 μL OPTI-MEM®-1 and then treatedwith 130 μL of the transfection mixture. Cells grown in 24-well platesor other standard tissue culture plates were treated similarly, usingappropriate volumes of medium and oligonucleotide. Cells were treatedand data were obtained in duplicate or triplicate. After approximately4-7 hours of treatment at 37° C., the medium containing the transfectionmixture was replaced with fresh culture medium. Cells were harvested16-24 hours after oligonucleotide treatment.

Control oligonucleotides are used to determine the optimal oligomericcompound concentration for a particular cell line. Furthermore, whenoligomeric compounds are tested in oligomeric compound screeningexperiments or phenotypic assays, control oligonucleotides are tested inparallel.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. The concentration ofpositive control oligonucleotide that results in 80% inhibition of thetarget mRNA is then utilized as the screening concentration for newoligonucleotides in subsequent experiments for that cell line. If 80%inhibition is not achieved, the lowest concentration of positive controloligonucleotide that results in 60% inhibition of the target mRNA isthen utilized as the oligonucleotide screening concentration insubsequent experiments for that cell line. If 60% inhibition is notachieved, that particular cell line is deemed as unsuitable foroligonucleotide transfection experiments. The concentrations ofantisense oligonucleotides used herein are from 50 nM to 300 nM when theantisense oligonucleotide is transfected using a liposome reagent and 1nM to 40 nM when the antisense oligonucleotide is transfected byelectroporation.

Example 2 Real-time Quantitative PCR Analysis of Target mRNA Levels

Quantitation of target mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM® 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured were evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. After isolation theRNA is subjected to sequential reverse transcriptase (RT) reaction andreal-time PCR, both of which are performed in the same well. RT and PCRreagents were obtained from Invitrogen Life Technologies (Carlsbad,Calif.). RT, real-time PCR was carried out in the same by adding 20 μLPCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each ofdATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-wellplates containing 30 μL total RNA solution (20-200 ng). The RT reactionwas carried out by incubation for 30 minutes at 48° C. Following a 10minute incubation at 95° C. to activate the PLATIN 9M® Taq, 40 cycles ofa two-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by RT, real-time PCR were normalizedusing either the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen® (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression was quantified by RT,real-time PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA was quantified using RiboGreen®RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). 170μL of RiboGreen® working reagent (RiboGreen® reagent diluted 1:350 in 10mM Tris-HCl, 1 mM EDTA, pH 7.5) was pipetted into a 96-well platecontaining 30 μL purified cellular RNA. The plate was read in aCytoFluor® 4000 (PE Applied Biosystems) with excitation at 485 nm andemission at 530 nm.

The GAPDH PCR probes have JOE covalently linked to the 5′ end and TAMRAor MGB covalently linked to the 3′ end, where JOE is the fluorescentreporter dye and TAMRA or MGB is the quencher dye. In some cell types,primers and probe designed to a GAPDH sequence from a different speciesare used to measure GAPDH expression. For example, a human GAPDH primerand probe set is used to measure GAPDH expression in monkey-derivedcells and cell lines.

Probes and primers for use in real-time PCR were designed to hybridizeto target nucleic acids using routine methods. For example,PrimerExpress® (Applied Biosystems, Foster City, Calif.) software isroutinely used to design probes and primers for use in real-time PCR.Examples of primer and probe sequences and the target nucleic acids towhich they hybridize are presented in Table 24. The target-specific PCRprobes have FAM covalently linked to the 5′ end and TAMRA or MGBcovalently linked to the 3′ end, where FAM is the fluorescent dye andTAMRA or MGB is the quencher dye.

TABLE 24 Target-specific primers and probes for use in real-time PCR SEQTarget Sequence ID Name Species Description Sequence (5′ to 3′) NO ApoBMouse Forward CGTGGGCTCCAGCATTCTA 1524 Primer ApoB Mouse ReverseAGTCATTTCTGCCTTTGCG 1525 Primer TC ApoB Mouse Probe CCAATGGTCGGGCACTGCT1526 CAA ApoB Mouse Forward GAAAATAGACTTCCTGAAT 1527 Primer AACTATGCATTApoB Mouse Reverse ACTCGCTTGCCAGCTTGC 1528 Primer ApoB Mouse ProbeTTTCTGAGTCCCCGTGCCC 1529 AACA GCGR Mouse Forward TGAGCCTTGCCACCTTCTC1530 Primer T GCGR Mouse Reverse GCGCACCCCAGCCAA 1531 Primer GCGR MouseProbe AGAGGAGCTTCTTTTCCCT 1532 CTACCTGGGC GCGR Mouse ForwardATTTCCTGCCCCTGGTACC 1533 Primer T GCGR Mouse Reverse CGGGCCCACACCT 1534Primer CTTG GCGR Mouse Probe CCACAAAGTGCAGCACC 1535 GCCTAGTGT PTEN MouseForward GCCACAGGCTCCCAGACAT 1536 Primer PTEN Mouse ReverseTCCATCCTCTTGATATCTC 1537 Primer CTTTTG PTEN Mouse ProbeACAGCCATCATCAAAGAGA 1538 TCGTTAGCAGAA PTEN Mouse ForwardATGACAATCATGTTGCAGC 1539 Primer AATTC PTEN Mouse ReverseCGATGCAATAAATATGCAC 1540 Primer AAATCA PTEN Mouse ProbeCTGTAAAGCTGGAAAGGGA 1541 CGGACTGGT

Example 3 Short Antisense Compounds Targeted to an ApoB Nucleic Acid andHaving 2′-MOE or Methyleneoxy (4′-CH₂—O-2′) BNA Modifications

Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected intraperitoneally (i.p.) with antisense compounds targeted toApoB, at a frequency of twice per week for three weeks. Antisensecompound doses included 2.4, 1.2, 0.6, 0.3 and 0.15 μmol/kg. Forantisense compounds 14 nucleotides in length, these doses equate toapproximately 12, 6, 3, 1.5 or 0.75 mg/kg, respectively. Shown in Table25 are the sequences and motifs of the antisense compounds used in thisstudy. The antisense compounds are either 20 or 14 nucleotides in lengthand have a central “gap” region consisting of ten 2′-deoxynucleotidesflanked by wings having 2′-O-methoxyethyl (2′-MOE) or BNA modified“wings.” For example, the 2-10-2 MOE gapmer motif indicates an antisensecompound with a gap of ten nucleotides flanked by 2 nucleotide wingswith 2′-MOE modifications. Bolded residues indicate 2′-O-methoxyethylmoieties and italicized residues indicate methyleneoxy (4′-CH₂—O-2′)BNAs. The internucleoside linkages of each compound are phosphorothioatethroughout. All cytosine residues of ISIS 147764 and ISIS 372938 arereplaced by 5-methyl cytosines. For ISIS 387462, only the cytosineresidue in the wing of the compound is replaced by 5-methyl cytosine.ApoB antisense compounds are targeted to publicly available ApoB-100sequences, including Genbank Accession No. XM_(—)137955.5 (SEQ ID NO:2).

TABLE 25 Antisense Compounds Targeted to an ApoB nucleic acid Target 5′SEQ ISIS SEQ Target Sequence ID NO ID NO Site (5′-3′) Gapmer Motif NO147764 2 8865 GTCCCTGAAGAT 5-10-5 MOE 1561 GTCAATGC 372938 2 8235GGTACATGGAAG 2-10-2 MOE 190 TC 387462 2 8235 GGTACATGGAAG 2-10-2 190 TCmethyleneoxy (4′-CH₂—O-2′) BNA

Forty-eight hours following the final injection, mice were sacrificed toevaluate transaminases (Table 26); liver and kidney weight (Table 27);triglyceride, LDL, HDL and free fatty acid levels (Table 28); targetmRNA level in liver (Table 29); target protein level in plasma; andoligonucleotide tissue concentration (Table 30). These endpoints weredetermined using methods described herein and well known to those ofordinary skill in the art.

TABLE 26 ALT and AST Levels (IU/L) Dose ISIS NO μmol/kg ALT AST SalineN/A 27.8 46.3 147764 2.4 29.5 64.0 372938 2.4 26.0 49.0 372938 1.2 24.849.5 372938 0.6 28.0 79.3 372938 0.3 28.3 60.0 372938 0.15 28.3 50.3387462 2.4 41.3 84.0 387462 1.2 35.3 63.5 387462 0.6 32.0 77.3 3874620.3 27.8 55.0 387462 0.15 29.3 68.3

TABLE 27 Liver and Kidney Weight (% of saline control) Dose ISIS NOμmol/kg Liver Kidney Saline N/A 100 100 147764 2.4 102 105 372938 2.4100 100 372938 1.2 90 101 372938 0.6 96 112 372938 0.3 91 107 3729380.15 96 98 387462 2.4 116 90 387462 1.2 113 90 387462 0.6 106 97 3874620.3 101 126 387462 0.15 95 100

Total body weight and food consumption did not differ significantlybetween saline-treated or oligonucleotide-treated animals. Glucoselevels also were similar among all treatment groups.

TABLE 28 Triglyceride (TRIG), Total Cholesterol (CHOL), HDL, LDL andFree Fatty Acid (FFA) Levels Dose TRIG CHOL HDL LDL FFA ISIS NO μmol/kg(mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) Saline N/A 167 107 81.8 11.01.76 147764 2.4 167 107 81.3 10.3 1.29 372938 2.4 153 104 79.0 10.3 1.28372938 1.2 136 101 77.8 9.5 1.70 372938 0.6 184 110 83.3 10.8 1.66372938 0.3 138 109 84.3 11.0 1.53 372938 0.15 151 106 82.8 10.8 1.57387462 2.4 49 14 9.0 1.5 0.74 387462 1.2 71 23 16.5 2.0 0.76 387462 0.6150 55 39.3 3.7 1.43 387462 0.3 136 92 72.8 7.5 1.14 387462 0.15 163 10481.5 9.3 1.47

TABLE 29 % ApoB mRNA Level (relative to saline control) 0.3 0.15 ISIS NO2.4 μmol/kg 1.2 μmol/kg 0.6 μmol/kg μmol/kg μmol/kg 147764 57.7 ND ND NDND 372938 77.0 90.0 87.3 92.6 93.1 387462 1.5 8.5 27.4 58.9 75.8

Treatment with ISIS 387462 resulted in a significant and dose-dependentdecrease in triglycerides, total cholesterol, HDL, LDL and free fattyacids. In accordance with these phenotypic findings, treatment with ISIS387462 also led to a dose-dependent reduction in ApoB mRNA (Table 29)and protein (not shown) levels in mouse plasma. To determine whether theobserved increase in efficiency with the methyleneoxy (4′-CH₂—O-2′) BNAgapmer is due to an increase in oligonucleotide accumulation,full-length and total oligonucleotide concentration in the liver andkidney were determined.

TABLE 30 Full-length and Total Antisense Compound Tissue Concentration(μM) Relative to ApoB mRNA level (% of saline control) Kidney Dose Full-Liver Kidney Liver ApoB ISIS NO μmol/kg Length Full-Length Total TotalmRNA 147764 2.4 28.6 22.9 33.5 31.3 58 372938 2.4 32.0 5.49 34.0 7.76 77387462 2.4 37.2 5.69 38.9 7.31 1.5 387462 1.2 29.8 3.71 31.3 4.91 8.5387462 0.6 18.9 1.97 20.0 2.57 27 387462 0.3 9.11 0.73 9.49 0.78 59387462 0.15 6.97 0.19 7.43 0.24 76

Levels of the 2-10-2 methyleneoxy (4′-CH₂—O-2′) BNA gapmer were similarto the 5-10-5 and 2-10-2 MOE gapmers in the kidney, but significantlyreduced in the liver. The EC₅₀ for ISIS 387462 in the liver wasdetermined by comparing oligonucleotide concentration in the liver toinhibition of ApoB mRNA. The approximate EC₅₀ for ISIS 387462 is 1 μM.In contrast, an effective 5-10-5 MOE gapmer compound typically has anEC₅₀ of approximately 15 μM in the liver.

Taken together, these results demonstrate that the ApoB short gapmerhaving methyleneoxy (4′-CH₂—O-2′) in the wings is a potent inhibitor oftarget mRNA expression and can effectively lower triglycerides,cholesterol and free fatty acids. The potency of the short antisensecompound does not appear to be a result of increased tissue accumulationsince similar levels of the compound were observed in kidney and reducedlevels were found in the liver, relative to the 5-10-5 MOE gapmer. Inaddition, the methyleneoxy (4′-CH₂—O-2′) BNA gapmer exhibited little tono adverse side effects.

Example 4 Short Antisense Compounds Targeted to a GCGR Nucleic Acid andHaving 2′-MOE Modifications

Eight-week old male C57/BL6 mice (Jackson Laboratory, Bar Harbor, Me.)were administered a single dose of GCGR oligonucleotide byintraperitoneal injection at a concentration of 6.25, 12.5, 25 or 50 mg.Each dose group consisted of four animals. Shown in Table 31 are thesequences, motifs and conjugates of the GCGR antisense compounds used inthis study. Bolded residues indicate 2′-O-methoxyethyl (2′-MOE)moieties. All compounds comprise phosphorothioate internucleosidelinkages throughout and each cytosine is replaced with 5-methylcytosine.ISIS 386626, ISIS 386627 and ISIS 386628 further comprise a C₁₆conjugate group attached to the 2′-O position of the sugar via a diamidelinkage (2′-OCH₂C(═O)N(H)(CH₂)₄N(H)C(═O)—(CH₂)₁₅CH₃). GCGR antisensecompounds target published GCGR sequences, including Genbank® AccessionNo. BC031885.1 (SEQ ID NO: 7).

TABLE 31 Short antisense compounds targeted to a GCGR nucleic acidTarget 5′ SEQ ISIS SEQ Target Gapmer ID NO ID NO Site Sequence (5′-3′)Motif Conjugate NO 148364 7 393 TGCACTTTGTGGTACCAAGG 5-10-5 MOE None1562 386626 7 1768 G_(C16) CTTCTCCATCATA 2-10-2 MOE C16 1563 386627 71244 G_(C16) GGCATGCTCGTCA 2-10-2 MOE C16 653 386593 7 1244GGGCATGCTCGTCA 2-10-2 MOE None 649 386628 7 1680 T_(C16) GTCTTGCTGCTTT2-10-2 MOE C16 1564 386594 7 1680 TGTCTTGCTGCTTT 2-10-2 MOE None 1565

Mice were sacrificed 48 hours following injection to determine serumtransaminase levels (Table 32); liver, white adipose tissue (WAT),spleen and kidney weight (Table 33); cholesterol, triglyceride andglucose levels (Table 34); GCGR mRNA levels (Tables 35-41); andfull-length and total oligonucleotide concentration in liver and kidney(Table 42). Endpoints were assessed using methods described herein andwell known to those of ordinary skill in the art. Data is included froma pre-treatment bleed (Pre-Bleed) and post-treatment bleed (Post-Bleed).

TABLE 32 ALT & AST Levels (IU/L) ISIS ALT ALT AST AST NO Dose (mg/kg)Pre-Bleed Post-Bleed Pre-Bleed Post-Bleed Saline N/A 36 51 55 85 14836450 24 40 40 115 148364 25 26 35 42 87 148364 12.5 23 32 44 69 1483646.25 28 34 47 76 386626 50 28 40 48 120 386626 25 30 36 44 92 38662612.5 28 34 44 90 386626 6.25 26 42 46 69 386627 50 27 457 42 451 38662725 29 97 45 142 386627 12.5 29 62 46 81 386627 6.25 23 87 38 96 38659350 23 33 46 58 386593 25 25 32 41 95 386593 12.5 26 33 43 74 386593 6.2528 31 43 53 386628 50 28 68 44 76 386628 25 24 32 40 57 386628 12.5 2835 42 75 386628 6.25 22 29 40 59 386594 50 29 34 46 92 386594 25 27 3147 82 386594 12.5 28 33 45 74 386594 6.25 23 48 42 67

TABLE 33 Organ Weights (% saline control) ISIS NO Dose (mg/kg) Liver WATKidney Spleen Saline N/A 100 100 100 100 148364 50 103 80 108 123 14836425 103 75 112 115 148364 12.5 100 84 108 96 148364 6.25 101 89 104 113386626 50 112 77 104 130 386626 25 109 97 103 120 386626 12.5 96 73 97114 386626 6.25 100 90 100 95 386627 50 90 113 102 165 386627 25 99 8799 143 386627 12.5 109 93 102 136 386627 6.25 103 96 102 131 386593 5096 98 102 118 386593 25 83 94 100 104 386593 12.5 99 82 101 129 3865936.25 96 77 98 144 386628 50 104 100 99 126 386628 25 102 97 109 113386628 12.5 101 111 99 114 386628 6.25 98 106 102 151 386594 50 90 80 99131 386594 25 93 76 99 128 386594 12.5 94 98 100 113 386594 6.25 102 85101 119

Overall, the GCGR antisense compounds exhibited little to no adverseside effects.

TABLE 34 Triglyceride (TRIG), Cholesterol (CHOL) and Glucose Levels(IU/L) TRIG TRIG CHOL CHOL Glucose Glucose ISIS NO Dose (mg/kg)Pre-Bleed Post-Bleed Pre-Bleed Post-Bleed Pre-Bleed Post-Bleed SalineN/A 132 181 91 96 208 285 148364 50 110 177 81 94 207 228 148364 25 115200 83 96 219 239 148364 12.5 106 179 85 89 198 256 148364 6.25 86 16286 89 226 215 386626 50 87 163 79 57 239 179 386626 25 100 187 87 72 235186 386626 12.5 100 148 82 76 232 185 386626 6.25 86 162 85 90 222 221386627 50 106 120 83 126 227 150 386627 25 101 148 90 115 218 203 38662712.5 99 203 86 98 237 219 386627 6.25 111 165 88 104 238 228 386593 50130 128 100 95 244 213 386593 25 119 135 83 77 206 208 386593 12.5 122128 83 79 222 233 386593 6.25 120 138 84 78 214 219 386628 50 102 98 8895 209 232 386628 25 102 129 84 85 210 223 386628 12.5 90 123 90 94 231240 386628 6.25 117 121 83 85 228 229 386594 50 93 99 84 85 203 274386594 25 106 94 90 86 219 272 386594 12.5 118 133 85 95 200 292 3865946.25 112 146 78 94 222 275

GCGR 2-10-2 MOE gapmers exhibited a trend toward lower post-bleedtriglyceride levels, relative to the 5-10-5 MOE gapmer, with ISIS 386628and ISIS 386594 having the greatest dose-dependent effect. Glucoselevels also were decreased in a dose-dependent manner followingtreatment with ISIS 386626 and ISIS 386627. Treatment with ISIS 386628,ISIS 386593 and ISIS 386594 also generally led to a decrease inpost-bleed glucose levels. Cholesterol levels did not appear tosignificantly differ among treatment groups.

To determine whether the phenotypic changes shown above correlated witha decrease in GCGR mRNA, treated animals were evaluated for levels oftarget mRNA in liver by real time PCR according to methods describedherein. Tables 35 to 41 show results from direct comparisons of theantisense compounds targeting GCGR nucleic acid for their effect ontarget expression. Results are expressed as percent of saline control.

TABLE 35 GCGR mRNA levels following treatment with ISIS 148364 & ISIS386626 ISIS NO 50 mg/kg 25 mg/kg 12.5 mg/kg 6.25 mg/kg 148364 36 79 8762 386626 0 8 3 7

TABLE 36 GCGR mRNA levels following treatment with ISIS 148364 & ISIS386627 ISIS NO 50 mg/kg 25 mg/kg 12.5 mg/kg 6.25 mg/kg 148364 63 87 10586 386627 3 30 57 74

TABLE 37 GCGR mRNA levels following treatment with ISIS 148364 & ISIS386593 ISIS NO 50 mg/kg 25 mg/kg 12.5 mg/kg 6.25 mg/kg 148364 56 74 10586 386593 9 38 74 90

TABLE 38 GCGR mRNA levels following treatment with ISIS 148364 & ISIS386628 ISIS NO 50 mg/kg 25 mg/kg 12.5 mg/kg 6.25 mg/kg 148364 42 77 98101 386628 2 18 53 77

TABLE 39 GCGR mRNA levels following treatment with ISIS 148364 & ISIS386594 ISIS NO 50 mg/kg 25 mg/kg 12.5 mg/kg 6.25 mg/kg 148364 59 98 10296 386594 25 47 50 96

TABLE 40 GCGR mRNA levels following treatment with ISIS 386627 & ISIS386593 ISIS NO 50 mg/kg 25 mg/kg 12.5 mg/kg 6.25 mg/kg 386627 5 40 58 42386593 10 29 34 71

TABLE 41 GCGR mRNA levels following treatment with ISIS 386628 & ISIS386594 ISIS NO 50 mg/kg 25 mg/kg 12.5 mg/kg 6.25 mg/kg 386628 4 13 38 97386594 19 50 56 99

Treatment with the 2-10-2 MOE gapmers led to a significantdose-dependent decrease in GCGR mRNA expression. ISIS 386626 exhibitedthe greatest decrease in target mRNA. To determine whether the observedincrease in efficiency with the short antisense compounds is due to anincrease in antisense compound accumulation, full-length and totalantisense compound concentration in the liver and kidney weredetermined.

TABLE 42 Total and Full-length Antisense Compound Concentrations inLiver and Kidney (μg/g) Full- Total Total Full-length length ISIS NOKidney Liver Kidney Liver 148364 90 54 58 46 386626 757 274 355 125386593 91 12 77 12 386628 496 286 305 202

The results shown in Table 42 demonstrate that short antisense compoundscomprising a C₁₆ conjugate exhibit a significant increase in antisensecompound accumulation in both liver and kidney. However, ISIS 386593,which was effective at reducing target mRNA, triglycerides and glucoselevels, accumulates to a level similar to the 5-10-5 MOE gapmer in liverand to a lower level in kidney. These results suggest that whileconjugation with C₁₆ can increase liver and kidney antisense compoundconcentration, it does not entirely account for the effectiveness of theshort antisense compounds.

Taken together, these results demonstrate that GCGR short antisensecompounds are capable of significantly inhibiting target mRNA expressionwhile also lowering triglyceride and glucose levels. In addition, withthe exception of ISIS 386627, the short MOE gapmers exhibited little tono toxic effects.

Example 5 Short Antisense Compounds Targeting to a GCGR Nucleic Acid andHaving 2′-MOE and Methyleneoxy (4′-CH₂—O-2′) BNA Modifications

Eight-week old male C57/BL6 mice (Jackson Laboratory, Bar Harbor, Me.)were administered a single dose of GCGR antisense compound byintraperitonel (i.p.) injection at a concentration of 10, 3.2, 1, and0.32 μmol·kg. Each dose group consisted of four animals. Shown in Table43 are the sequences, motifs and conjugates of the GCGR antisensecompounds used in this study. Bolded residues indicate 2′-O-methoxyethyl(2′-MOE) modifications and the italicized residues indicate methyleneoxy(4′-CH₂—O-2′) BNA modifications. All antisense compounds comprisephosphorothioate internucleoside linkages throughout and each cytosineis replaced with 5-methylcytosine. GCGR antisense compounds targetpublished GCGR nucleic acids, including Genbank Accession No. BC031885.1(SEQ ID NO: 7).

TABLE 43 Antisense Compounds targeted to a GCGR nucleic acid Target 5′SEQ ISIS SEQ ID Target Sequence ID NO NO Site (5′-3′) Gapmer Motif NO148364 7 393 TGCACTTTGTGGT 5-10-5 MOE 1562 ACCAAGG 396144 7 1768GCTTCTCCATCA 2-10-2 MOE 1566 TA 396148 7 1768 GCTTCTCCATCA 2-10-2 1567TA Methyleneoxy (4′-CH₂—O-2′) BNA 396145 7 1765 ATGGCTTCTCCAT 5-10-5 MOE1568 CATATCC 396146 7 1244 GGGCATGCTCG 2-10-2 MOE 650 TCA 396149 7 1244GGGCATGCTCGT 2-10-2 652 CA Methyleneoxy (4′-CH₂—O-2′) BNA 396147 7 1241CTTGGGCATGCTC 5-10-5 MOE 1569 GTCAGTC

To determine whether the phenotypic changes shown above correlated witha decrease in GCGR mRNA, treated animals were evaluated for levels oftarget mRNA in liver by RT, real time PCR according to methods describedherein. Table 44 show results from direct comparisons of the antisensecompounds targeting GCGR nucleic acid for their effect on targetexpression. Results are expressed as percent of saline control.

TABLE 44 GCGR mRNA levels ISIS NO. 0.32 μmol/kg 1 μmol/kg 3.2 μmol/kg 10μmol/kg 148364 105  106 73 38 396144 122  117 40 35 396148 20  6 2 1396145 nd Nd 33 8 396146 98 135 95 35 396149 91  41 30 7 396147 nd Nd 6828

As shown in Table 44, each short antisense compound having methyleneoxy(4′-CH₂—O-2′) BNA modifications demonstrated a dose-dependent reductionin GCGR mRNA levels. Furthermore, the short antisense compounds weremore effective at target reduction than the 5-10-5 MOE gapmer. Eachshort antisense compound comprising methyleneoxy (4′-CH₂—O-2′) BNA inthe wings resulted in a significant reduction in GCGR protein relativeto both saline control and ISIS 148364 treatment. Next, estimated ED₅₀concentrations for each antisense were calculated using Graphpad Prism;ED₅₀ is the dose at which 50% mRNA reduction is observed. The resultsare shown below in Table 45.

TABLE 45 Estimated ED₅₀ Concentration ISIS Gapmer Motif NO ED₅₀(μmole/kg) ED₅₀ (mg/kg) 5-10-5 MOE 148364 7 50.6 2-10-2 MOE 396144 418.1 2-10-2 methyleneoxy BNA 396148 0.1 0.4 5-10-5 MOE 396145 2.1 9.32-10-2 MOE 396146 8.3 40 2-10-2 methylenexy BNA 396149 1.1 5 5-10-5 MOE396147 5.2 37.5

Example 6 Short Antisense Compounds Targeting a PTEN Nucleic Acid andHaving Methyleneoxy (4′-CH₂—O-2′) BNA Modifications

Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereadministered a single i.p. injection of PTEN antisense compound at adose of 8 μmol/kg. Each dose group consisted of four animals. Shown inTable 46 are the sequences and motifs of the PTEN antisense compoundsused in this study. Bolded residues indicate 2′-O-methoxyethyl moieties(2′-MOE) and italicized residues indicate Methyleneoxy BNA nucleotides.Each antisense compound comprises phosphorothioate linkages throughout.In addition, the cytosine residues in the gap of ISIS 384073 and in thewings of ISIS 392056, ISIS 392057, ISIS 392061 and ISIS 392063 arereplaced with 5-methylcytosines. Antisense compounds target publishedPTEN nucleic acids, including Genbank Accession No. U92437.1 (SEQ ID

TABLE 46 Antisense Compounds targeted to a PTEN nucleic acid Target 5′ISIS SEQ ID Target SEQ NO NO Site Sequence (5′-3′) Gapmer Motif ID NO141923 Control N/A CCTTCCCTGAAGGTTCCTCC 5-10-5 MOE 1570 116847 29 2011TCAAATCCAGAGGCTAGCAG 5-10-5 MOE 1571 384073 29 2013 AAATCCAGAGGCTAGC3-10-3 methyleneoxy 1428 (4′-CH₂—O-2′) BNA 391172 29 2013AAATCCAGAGGCTAG 2-10-3 methyleneoxy 1429 (4′-CH₂—O-2′) BNA 392056 29 140AGCTGCAGCCATGA 2-10-2 methyleneoxy 1263 (4′-CH₂—O-2′) BNA 392057 29 807GGTCCAGGGCCAAG 2-10-2 methyleneoxy 1162 (4′-CH₂—O-2′) BNA 392061 29 2014AATCCAGAGGCTAG 2-10-2 methyleneoxy 1431 (4′-CH₂—O-2′) BNA 392063 29 3099AGGCCAGTGCTAAG 2-10-2 methyleneoxy 1226 (4′-CH₂—O-2′) BNA

Mice were sacrificed 72 hours following injection to determine serumtransaminase levels (Table 47); liver and spleen weights (Table 47); andPTEN mRNA levels in liver, kidney and fat (Table 48), according toprocedures described herein and well know to one of ordinary skill inthe art.

TABLE 47 Transaminase Levels and Organ Weights Liver ISIS AST ALT WeightSpleen Weight NO (IU/L) (IU/L) % Saline % Saline Saline 98.5 37.5 100100 141923 89.5 34.8 101 108 116847 59.8 29.5 109 108 384073 57.8 29.3115 111 391172 48.5 32.8 120 112 392056 516 892 125 167 392057 63.8 34.5125 101 392061 189 42.0 123 111 392063 67.3 21.8 127 134

Overall, the short antisense compounds with methyleneoxy (4′-CH₂—O-2′)BNA modifications exhibited little to no adverse effects. In addition,total body weight did not significantly differ between treatment groups.

TABLE 48 % PTEN mRNA levels in Liver, Kidney and Fat ISIS NO LiverKidney Fat Saline 100 100 100 141923 102 133 118 116847 37 96 85 38407324 74 77 391172 18 63 101 392056 27 88 74 392057 33 79 96 392061 24 6185 392063 6.5 52 72

As shown in Table 48, each antisense compound targeted to a PTEN nucleicacid led to a significant reduction in target mRNA levels in liver ascompared with saline treated and control treated animals. The antisensecompounds had various effects on target mRNA levels in kidney and fat.

Example 7 Short Antisense Compounds Targeting a PTEN nucleic acid andhaving BNA Modifications

Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereadministered a single intraperitoneal (i.p.) injection of antisensecompound targeted a PTEN nucleic acid at a dose of 8, 4, 2 or 1 μmol/kg.Each dose group consisted of four animals. Shown in Table 49 are thesequence, wing chemistry and motif of each antisense compound used inthis study. Bold residues indicate 2′-MOE modified nucleotides,italicized letters indicate methyleneoxy (4′-CH₂—O-2′) BNAmodifications. All antisense compounds comprise phosphorothioatelinkages at each position. Each cytosine of ISIS 116847 and the cytosineresidues in the methyleneoxy (4′-CH₂—O-2′) BNA wings of ISIS 392063 arereplaced with 5-methylcytosines, while the thymidine residues in themethyleneoxy (4′-CH₂—O-2′) BNA wings of ISIS 392745 are replaced with5-methyl thymidines. Antisense compounds target published PTEN nucleicacids, including Genbank Accession No. U92437.1 (SEQ ID NO: 13).

TABLE 49 Antisense Compounds Targeted to a PTEN Nucleic Acid Target SEQISIS SEQ Target Sequence ID NO ID NO Site (5′-3′) Gapmer Motif NO 11684713 2011 TCAAATCCAGAGG 5-10-5 1571 CTAGCAG MOE 392063 13 3099CTTAGCACTGGC 2-10-2 1226 CT Methyleneoxy BNA 392745 13 3099 CTTAGCACTGGC2-10-2 1226 CT methyleneoxy BNA

Mice were sacrificed 72 hours following injection to determine serumtransaminase levels (Table 50); liver, kidney and spleen weights (Table50); PTEN mRNA levels in liver (Table 51); and estimated ED₅₀oligonucleotide concentration (Table 52). These endpoints were measuredusing methods described herein and well known to those of ordinary skillin the art.

TABLE 50 AST, ALT and Bilirubin Levels and Organ Weights Liver KidneySpleen Dose AST ALT Bilirubin Weight % Weight Weight ISIS NO μmol/kg(IU/L) (IU/L) (mg/dL) Saline % Saline % Saline Saline N/A 64.0 31.8 0.15100 100 100 116847 8 73.0 32.0 0.1 114 92 106 392063 8 50.3 17.3 0.1 11598 115 392063 4 100.8 31.3 0.15 122 94 116 392063 2 60.5 32.8 0.1 112 99106 392063 1 57.5 29.3 0.1 104 95 107 392745 8 75.5 23.5 0.13 125 99 100392745 4 77.0 29.3 0.13 121 100 96 392745 2 69.0 32.0 0.13 110 98 103392745 1 52.0 27.3 0.1 109 97 104

Overall, the PTEN antisense compounds did not show significant signs oftoxicity. Kidney, liver and spleen weights were all within normalranges. Total body weight did not significantly differ between treatmentgroups.

TABLE 51 % PTEN mRNA levels in Liver (relative to saline control) ISISNO 8 μmol/kg 4 μmol/kg 2 μmol/kg 1 μmol/kg 116847 36 ND ND ND 392063 7.416 32 60 392745 5.2 11 31 60

As shown in Table 51, each short antisense compound having methyleneoxy(4′-CH₂—O-2′) BNA modifications demonstrated a dose-dependent reductionin PTEN mRNA levels. Furthermore, the short antisense compounds weremore effective at target reduction than the 5-10-5 MOE gapmer. Levels ofPTEN protein in liver were also determined following administration ofeach antisense compound at a dose of 8 μmol/kg. Each short antisensecompound comprising methyleneoxy (4′-CH₂—O-2′) BNA in the wings resultedin a significant reduction in PTEN protein relative to both salinecontrol and ISIS 116847 treatment. Next, estimated ED₅₀ concentrationsfor each oligonucleotide were calculated using Graphpad Prism. Theresults are shown below in Table 52.

TABLE 52 Estimated ED₅₀ Concentration ISIS Wing Chemistry NO ED₅₀(μmole/kg) ED₅₀ (mg/kg) MOE (with 5-MeC) 116847 6.3 45.2 methyleneoxyBNA 392063 1.3 5.8 (with 5-MeC) methyleneoxy BNA 392745 1.2 5.6

To further investigate different types of bicyclic nucleic acidcompounds, an additional set of short antisense compounds targeting aPTEN nucleic acid was designed and tested. Six-week old male Balb/c mice(Jackson Laboratory, Bar Harbor, Me.) were administered a singleintraperitoneal (i.p.) injection of antisense compound at a dose of 8,4, 2 or 1 μmol/kg. Each dose group consisted of four animals. Shown inTable 53 are the sequence, wing chemistry and motif of each antisensecompound used in this study. All antisense compounds comprisephosphorothioate linkages at each position. The cytosine residues in themethyleneoxy (4′-CH₂—O-2′) BNA wings of ISIS 392063 are replaced with5-methylcytosines. The antisense compound target published PTEN nucleicacids, including Genbank Accession No. U92437.1 (SEQ ID NO: 13).

TABLE 53 Antisense Compounds Targeting a PTEN Nucleic Acid Target 5′ SEQISIS SEQ Target Sequence ID NO ID NO Site (5′-3′) Gapmer Motif NO 39206329 3099 CTTAGCACT 2-10-2 1226 GGCCT Methyleneoxy BNA 396564 29 3099CTTAGCACT 2-10-2 1226 GGCCT Oxyamino (4′-CH₂—N(R)—O- 2′) BNA 396006 293099 CTTAGCACT 2-10-2α-L- 1226 GGCCT Methyleneoxy BNA

Mice were sacrificed 72 hours following injection to determine serumtransaminase levels (Table 54); liver and spleen weights (Table 54); andPTEN mRNA levels in liver (Table 55), according to methods describedherein and well known to those of ordinary skill in the art.

TABLE 54 AST and ALT Levels and Organ Weights ISIS Dose AST ALT LiverSpleen NO μmol/kg (IU/L) (IU/L) Weight Weight Saline N/A 71 33 100 100392063 8 97 38 118 103 392063 4 179 36 115 107 392063 2 67 32 109 116392063 1 68 27 102 105 396564 8 67 25 100 104 396564 4 96 30 102 106396564 2 68 27 100 119 396564 1 79 39 97 109 396006 8 56 28 110 104396006 2 139 36 97 105

TABLE 55 % PTEN mRNA levels in Liver (relative to saline control) ISISNO 8 μmol/kg 4 μmol/kg 2 μmol/kg 1 μmol/kg 392063 6.9 18  39 71 39656486 97 100 96 396006 6.5 ND ND 70

As shown above, short antisense compounds having α-L-methyleneoxy(4′-CH₂—O-2′) BNA modifications led to a dose-dependent reduction intarget mRNA levels. Treatment with the short antisense compound havingoxyamino BNA modifications led to a modest reduction in targetexpression.

Example 8 Single Dose Administration Dose Response Study with ShortAntisense Compounds Targeting ApoB and PTEN nucleic acids

Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereadministered a single intraperitoneal (i.p.) injection of antisensecompound at a dose of 8, 4, 2 or 1 μmol/kg. Each dose group consisted offour animals. Shown in Table 56 are the sequence, wing chemistry andmotif of each antisense compound used in this study. Italicized residuesindicate methyleneoxy (4′-CH₂—O-2′) BNA modifications, underlinedresidues indicate N-methyl-oxyamino (4′-CH₂—N(CH₃)—O-2′) BNAmodifications, and boxed residues indicate α-L-methyleneoxy(4′-CH₂—O-2′) BNA modifications. All antisense compounds comprisephosphorothioate linkages at each position. Each cytosine of ISIS 116847and the cytosine residues in the methyleneoxy (4′-CH₂—O-2′) BNA wings ofISIS 392063 are replaced with 5-methylcytosines, while the thymidineresidues in the methyleneoxy (4′-CH₂—O-2′) BNA wings of ISIS 392745 arereplaced with 5-methyl thymidines. PTEN antisense compounds targetpublished PTEN nucleic acid, including Genbank Accession No. U92437.1(SEQ ID NO: 13). ApoB antisense compounds target published ApoB nucleicacid, including Genbank Accession No. XM-137955.5 (SEQ ID NO: 2).

TABLE 56 Short Antisense Compounds Targeted to ApoB and PTEN NucleicAcids ISIS Target 5′ Target SEQ ID NO Target Seq ID Site SEQUENCE GapmerNO 387462 ApoB 19 8235 GGTACATGGAAGTC 2-10-2 193 Methyleneoxy BNA 392063PTEN 29 3099 CTTAGCACTGGCCT 2-10-2 1226 Methyleneoxy BNA 396565 PTEN 293099 CUTAGCACTGGCCU 2-10-2 1226 N-Me-oxyamino BNA 396006 PTEN 29 3099CUTAGCACTGGCCU 2-10-2 1226 α-L-methyleneoxy BNA

TABLE 57 % ApoB and PTEN mRNA Reduction (relative to saline control) %ApoB ISIS Dose mRNA Reduction % PTEN mRNA Reduction NO (μmol/kg)(relative to saline) (relative to saline) 387462 8 0.62 92.8 4 6.55 1032 18.6 105 1 42.0 98.0 392063 8 126 6.79 4 111 18.1 2 112 42.4 1 11462.3 396565 8 116 23.8 4 1.04 46.6 2 94.4 76.1 1 115 89.5 396006 8 94.362.9 4 101 18.2 2 79.7 52.4 1 111 82.4

As shown in Table 57, each short antisense compound having MethyleneoxyBNA modifications demonstrated a dose-dependent reduction in target mRNAlevels. Notably, the short antisense compound with N-methyl-oxyamino BNAwings (ISIS 396565) also demonstrated dose-dependent reduction in PTENexpression similar to both the β-D-methyleneoxy BNA and α-L-methyleneoxyBNA short antisense compounds. Next, estimated ED₅₀ concentrations foreach antisense were calculated using Graphpad Prism. The results areshown below in Table 58.

TABLE 58 Estimated ED₅₀ Concentrations ISIS Wing Chemistry NO ED₅₀(μmole/kg) ED₅₀ (mg/kg) Methyleneoxy BNA 387462 0.8 3.9 Methyleneoxy BNA392063 1.5 7 N-Me-oxyamino BNA 396565 3.8 17.4 α-L-methyleneoxy BNA396006 2.1 9.3

Example 9 Administration of a Parent and Parent Mixed Backbone AntisenseCompound Targeting SGLT-2 mRNA

ISIS 257016 was administered to db/db mice (Charles River Laboratories,Wilmington, Mass.) intraperitoneally at a dose of 1, 7.5, 14 or 17 mg/kgtwice a week. Control groups included a group receiving saline on thesame dosing schedule and a group receiving ISIS 145733. ISIS 257016 andISIS 145733 both comprise the sequence GAAGTAGCCACCAACTGTGC (SEQ ID NO:1572) further comprising a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. All cytidine residues are5-methylcytidines. The internucleoside (backbone) linkages arephosphorothioate (P═S) throughout the oligonucleotide for ISIS 145733;however ISIS 257016 has a mixed backbone. The internucleoside linkagesfor ISIS 257016 are phosphodiester (P═O) in the wings andphosphorothioate in the gap. Forty-eight hours following administrationof the last dose the mice were sacrificed and kidney tissue was analyzedfor SGLT-2 mRNA levels. The results are shown below in Table 59.

TABLE 59 Antisense inhibition of SGLT2 mRNA expression in vivo by 5-10-5MOE gapmers % change in SGLT2 expression Dose of oligonucleotiderelative to saline nmol/kg ISIS 145733 ISIS 257016 17 −37.5 −76 14−31.25 −74 7.5 −12.5 −62.5 1 +3 −44

Both ISIS 257016 and ISIS 145733 markedly reduced SGLT-2 levels comparedto saline control. (mRNA levels determined using RT, real-time PCR asdescribed above) However, ISIS 257016 has been shown to be about 20-50times more potent for reducing SGLT-2 mRNA compared to ISIS 145733. Anassociated reduction in plasma glucose levels was seen for the treatmentgroups (661±14 for the saline group compared to 470±23 for the groupreceiving ISIS 257016). Accumulation of ISIS 257016 and ISIS 145733 inthe kidney was similar over the dose range, however little of the fulllength 257016 antisense was detected in the kidney which supports thetheory that a degradation product is responsible for the increasedactivity. Also the onset of action following a single dose of 25 mg/kgcorrelated to a time pint were little intact 257016 antisense compoundwas left.

Similar studies were performed in lean mice, ob/ob mice and in ZDF rats(Charles Rivers Laboratories) using ISIS 257016, ISIS 145733 or salinein a similar same dosing schedule as described above. The sequence ofthe binding site for ISIS 145733 and ISIS 257016 is conserved betweenmouse and rat (see Table 60). Reduction of SGLT-2 mRNA in the kidney wassimilar to that seen above. In a study utilizing rats, at a dose of 10mg/kg given two times a week for two weeks, ISIS 145733 was shown toreduce SGLT-2 mRNA levels by about 40% whereas the reduction achievedwith ISIS 257016 was greater than 80%. ISIS 257016 reduces SGLT2expression maximally at a low dose of 12.5 mg/kg. Additional studies atlower dosing ranges show significant reduction of SGLT2 mRNA levels withthe mixed backbone antisense compound at doses less than 1 mg/kg/wk.

Example 10 Administration of a Parent and Short Antisense CompoundTargeting SGLT-2 mRNA

Pharmacokinetic studies indicated that ISIS 257016 was acting as aprodrug that was metabolized to a 12 nucleobase pharmacophore. In a nextstudy, ZDF rats were dosed intraperitoneally twice per week with 1.5mg/kg of either ISIS 257016 or ISIS 370717, or with saline at a similardosing schedule. ISIS 370717 is a 12 nucleobase antisense compoundtargeted to SGLT-2 nucleic acid comprising the sequence TAGCCACCAACT(SEQ ID NO: 154) and further comprising central “gap” region consistingof ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by one-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. All cytidine residues are5-methylcytidines. The internucleoside (backbone) linkages arephosphorothioate (P═S) throughout the oligonucleotide.

Following five weeks of dosing the animals were sacrificed and kidneytissue was analyzed for SGLT-2 mRNA levels. The pharmacological activityof ISIS 257016 and ISIS 370717 were similar, however, the 12 nucleotideantisense compound displayed a faster onset of action. ISIS 370717displayed nearly 80% inhibition of SGLT2 expression in kidney on day twoafter a single dose of 2.8 umoles/kg whereas ISIS 257016 displayed onlyabout 25% inhibition on day 2 after the same single dose administration.The date support that ISIS 257016 is a prodrug having a 12 nucleotidepharmacophore.

Example 11 Potency and Bioavailability of a Short Antisense Compound

The improved potency displayed by ISIS 370717 and the improved oralbioavailability for these short antisense compounds makes thesecompounds useful for oral administration. Normal rats received ISIS370717, ISIS 145733 or saline at 100 mg/kg twice per week viaintrajejunal administration. About 48 hours following the last dose, theanimals were sacrificed and kidney tissue was analyzed for antisensecompound concentration and SGLT-2 mRNA levels. There was a significantlyhigher accumulation of ISIS 370717 in the kidney tissue (approximately500 micro grams per gram of tissue) compared to the controls. Moreover,SGLT-2 mRNA was reduced by more than 80% over the controls.

Example 12 Wing, Gap and Total Length Variations Around a 12 NucleotideShort Antisense Compound

ISIS 3707171-10-1 MOE gapmer was used as a template to make sequencerelated oligos with varying motifs. These variations are provided inTable 60. The antisense compounds were designed to target differentregions of the mouse or rat SGLT2 nucleic acid, using publishedsequences (GenBank accession number U29881.1, incorporated herein as SEQID NO: 1575, and GenBank accession number AJ292928.1, incorporatedherein as SEQ ID NO: 1576, respectively).

TABLE 60 Short Antisense compounds targeting SGLT2 nucleic acids5′ Target Site 5′ Target Site on mouse on rat SEQ ISIS SEQ ID NO: SEQ IDNO: Gapmer ID NO 1575 1576 Motif Sequence (5′-3′) NO 257016 2680 1485-10-5 GAAGTAGCCACCAACTGTGC 1553 MOE 370717 2684 152 1-10-1 TAGCCACCAACT1554 MOE 386169 2684 152 2-8-2 TAGCCACCAACT 1555 MOE 386176 2685 1531-8-1 AGCCACCAAC 1556 MOE 386196 2684 152 3-6-3 TAGCCACCAACT 1557 MOE

The antisense compounds were analyzed for their effect on mouse SGLT2mRNA levels. Data are ranges taken from three experiments in which micewere dosed twice per week for three weeks with 2.5, 0.5 or 0.1 umol/kgof the above MOE gapmers given by intraperitoneal injection. Mice weresacrificed 48 hours following last administration and evaluated forSGLT2 levels in kidney. SGLT2 mRNA levels were determined by RT,real-time PCR as described by other examples herein. PCR results werenormalized to an internal ISIS control. The results are shown below inTable 61.

TABLE 61 Antisense inhibition of SGLT2 in vivo by 1-10-1 and 1-10-2 MOEgapmers % change in SGLT2 expression relative to saline Dose ofoligonucleotide ISIS ISIS ISIS ISIS ISIS umol/kg 370717 386169 386176386196 386197 2.5 −82 −85 −80 −50 −20 0.5 −70 −80 −68 −30 −15 0.1 −55−70 −65 −35 −20

These results illustrate that all the various motifs tested inhibit theexpression of SGLT2 in vivo in a dose-dependent manner. The 1-10-1,2-8-2 and 1-8-1 gapmers were found to be particularly potent.

Example 13 Antisense Inhibition of Rat SGLT-2 by 1-10-1 and 1-10-2 MOEGapmers

1-10-1 and 1-10-2 MOE gapmer antisense compounds, provided in Table 62,were designed to target different regions of the mouse or rat SGLT2 RNA.All short antisense compounds in Table 62 are chimeric oligonucleotides(“gapmers”) either 12 or 13 nucleotides in length, composed of a central“gap” segment consisting of ten 2′-deoxynucleotides, which are flankedon the 5′ side by a one-nucleoside “wing” and on the 3′ side by a two orone-nucleotide “wing”. The wings are composed of 2′-methoxyethyl(2′-MOE) nucleotides. The internucleoside (backbone) linkages arephosphorothioate (P═S) throughout the oligonucleotide. All cytidineresidues are 5-methylcytidines.

TABLE 62 Antisense compounds targeting SGLT2 nucleic acid 5′ Target Site5′ Target Site on SEQ ID on SEQ ID NO: XXX NO: XXX Gapmer SEQ ISIS NO(mouse) (rat) Motif Sequence (5′-3′) ID NO 370717 2684 152 1-10-1 MOETAGCCACCAACT 1554 382675 2683 151 1-10-1 MOE TAGCCACCAACTG 1559 379692508 1-10-1 MOE TGTTCCAGCCCA 246 382676 507 1-10-2 MOE TGTTCCAGCCCAG 246379699 1112 1-10-2 MOE GGCATGAGCTTC 281 382677 1111 1-10-2 MOEGGCATGAGCTTCA 281 382677 958 1-10-2 MOE GGCATGAGCTTCA 281

The short antisense compounds were analyzed for their effect on ratSGLT2 mRNA levels. Data are ranges taken from three experiments in whichMale Sprague-Dawley rats (170-200 g) were dosed twice per week for threeweeks with 450, 150 or 50 nmol/kg of either a 1-10-1 or 1-10-2 MOEgapmer given by intraperitoneal injection. Rats were sacrificed 48 hoursfollowing last administration and evaluated for SGLT2 mRNA levels inkidney. Target levels were determined by RT, real-time PCR as describedby other examples herein. PCR results were normalized to an internalISIS control. The results are shown below in Table 63.

TABLE 63 Antisense inhibition of SGLT2 mRNA in vivo by 1-10-1 and 1-10-2MOE gapmers % change in SGLT2 expression relative to saline Dose of ISISISIS ISIS ISIS ISIS ISIS oligonucleotide 370717 382675 379692 382676379699 382677 nmol/kg 1-10-1 1-10-2 1-10-1 1-10-2 1-10-1 1-10-2 450 −70−80 −90 −85 −83 −75 150 −70 −65 −85 −80 −75 −60 50 −55 −50 −80 −65 −60−40

These results illustrate that both the 1-10-1 and 1-10-2 MOE gapmersreduce SGLT2 mRNA in vivo in a dose-dependent manner.

Rats were further evaluated for total body weight, liver, spleen andkidney weight. Significant changes in spleen, liver or body weight canindicate that a particular compound causes toxic effects. All changeswere within the margin of error of the experiment. No significantchanges in body weight were observed during the treatment or at studytermination. No significant changes in liver or spleen weights wereobserved.

Toxic effects of short antisense compounds administered in vivo can alsobe assessed by measuring the levels of enzymes and proteins associatedwith disease or injury of the liver or kidney. Elevations in the levelsof the serum transaminases aspartate aminotransferase (AST) and alanineaminotransferase (ALT) are often indicators of liver disease or injury.Serum total bilirubin is an indicator of liver and biliary function, andalbumin and blood urea nitrogen (BUN) are indicators of renal function.Glucose and triglyceride levels are sometimes altered due to toxicity ofa treatment. Serum glucose also depends in part upon the activity ofSGLT2. The levels of ALT, AST, total bilirubin, albumin, BUN, glucoseand triglyceride were measured in rats treated with the short antisensecompounds. The levels of routine clinical indicators of liver and kidneyinjury and disease were within normal ranges and are not significantlychanged relative to saline-treated animals, demonstrating that the shortantisense compounds do not significantly affect renal or hepaticfunction. Triglyceride and glucose levels were not significantlyelevated relative to saline-treated animals.

Example 14 Antisense Inhibition of Mouse and Rat SGLT2 by 1-10-1 MOEGapmers

1-10-1 MOE gapmer antisense compounds designed to target differentregions of mouse SGLT2 mRNA are shown in Table 64.

TABLE 64 Composition of Antisense Compounds Targeting SGLT2 mRNA5′ Target Site 5′ Target Site on SEQ ID on SEQ ID ISIS NO: XXX NO: XXXSEQ NO (mouse) (rat) Motif Sequence (5′-3′) ID NO 370717 2684 152 1-10-1MOE TAGCCACCAACT 1554 379692  508 1-10-1 MOE TGTTCCAGCCCA 246 3796991112 1-10-1 MOE GGCATGAGCTTC 281 379702 1525 1-10-1 MOE GCACACAGCTGC 293381408   3034** 1-10-1 MOE TACCGAACACCT 1560 **indicates 3 mismatches toa target sequence

The short antisense compounds were analyzed for their effect on mouseSGLT2 mRNA levels. Data was taken from three experiments in which Male6-week old Balb/c mice were dosed twice per week for two weeks with 450,150, or 50 nmol/kg of one of the above 1-10-1 MOE gapmers given byintraperitoneal injection. Mice were sacrificed 48 hours following lastadministration and evaluated for SGLT2 mRNA levels in kidney. Targetlevels were determined by RT, real-time PCR as described by otherexamples herein. PCR results were normalized to an internal ISIScontrol. The results are shown below in Table 65.

TABLE 65 Antisense inhibition of SGLT2 mRNA in vivo by 1-10-1 MOEgapmers % change in SGLT2 expression relative to saline Dose ofoligonucleotide ISIS ISIS ISIS ISIS ISIS nmol/kg 370717 379692 379699379702 381408 450 −65 −80 −80 −75 — 150 −55 −70 −62.5 −72.5 — 50 −47.5−52.5 −42.5 −52.5 —

These results illustrate that all the 1-10-1 MOE gapmers except, ISIS381408, inhibit the expression of SGLT2 in vivo in a dose-dependentmanner in mouse. Activity of ISIS 381408 has been shown in Rat studies(See Table 65).

Evaluation of 1-10-1 Gapmers in Rat

The effect of the above 1-10-1 gapmers (see Table 64 above) on rat SGLT2mRNA levels. Data are taken from four experiments in which maleSprague-Dawley rats (170-200 g) were dosed twice per week for threeweeks with 250 nmol/kg given by intraperitoneal injection. Rats weresacrificed 48 hours following last administration and evaluated forSGLT2 mRNA levels in kidney. Target levels were determined by RT,real-time PCR as described by other examples herein. PCR results werenormalized to an internal ISIS control. The results are shown below inTable 66.

TABLE 66 Antisense inhibition of SGLT2 mRNA in vivo by 1-10-1 MOEgapmers % change in SGLT2 expression relative to saline Dose ofoligonucleotide ISIS ISIS ISIS ISIS ISIS nmol/kg 370717 379692 379699379702 381408 250 −70 −85 −75 −25 −5

These results illustrate that all the 1-10-1 MOE gapmers inhibit theexpression of SGLT2 in in vivo rat studies.

Example 15 Antisense Inhibition of Mouse and Rat SGLT2 Expression byAdditional 1-10-1 and 2-8-2 MOE Gapmers

1-10-1 and 2-8-2 MOE gapmer short antisense compounds were designed totarget different regions of the mouse SGLT2 RNA but have complementarityacross species. The short antisense compounds are shown in Table 67. Allshort antisense compounds in Table 67 are gapmers 12 nucleotides inlength, composed of a central “gap” segment consisting of2′-deoxynucleotides, which are flanked on both sides (5′ and 3′directions) by wing segments having 2′-modifications. The wings arecomposed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. All cytidine residues are 5-methylcytidines.

TABLE 67 Short Antisense Compounds Targeting SGLT2 nucleic acid 5′Target Target SEQ ID Gapmer SEQ ID ISIS NO Site (rat) (rat) MotifSequence (5′-3′) NO 379692 508 1-10-1 MOE TGTTCCAGCCCA 246 388625 5081-10-1 MOE TGTTCCAGCCCA 246 379699 1112 1-10-1 MOE GGCATGAGCTTC 281388626 1112 2-8-2 MOE GGCATGAGCTTC 281 379702 1525 2-8-2 MOEGCACACAGCTGC 293 388627 1525 2-8-2 MOE GCACACAGCTGC 293

The short antisense compounds were analyzed for their effect on mouseSGLT2 mRNA levels in vivo. Data was taken from three experiments inwhich male 6-week old Balb/c mice were dosed twice per week for threeweeks with 0.5, 0.1, or 0.02 umol/kg of either a 1-10-1 or 2-8-2 MOEgapmer given by intraperitoneal injection. Mice were sacrificed 48 hoursfollowing last administration and evaluated for SGLT2 levels in kidney.Target levels were determined by RT, real-time PCR as described by otherexamples herein. PCR results were normalized to an internal ISIScontrol. The results are shown below in Table 68.

TABLE 68 Antisense inhibition of SGLT2 mRNA in vivo by 1-10-1 and 2-8-2MOE gapmers % change in SGLT2 expression relative to saline Dose of ISISISIS ISIS ISIS ISIS ISIS oligonucleotide 379692 388625 379699 388626379702 388627 umol/kg 1-10-1 2-8-2 1-10-1 2-8-2 1-10-1 2-8-2 0.5 −85 −90−75 −80 −70 −65 0.1 −75 −88 −60 −60 −65 −50 0.02 −55 −65 −30 −45 −40 −38

These results illustrate that both the 1-10-1 and 2-8-2 MOE gapmersinhibit the expression of SGLT2 in vivo in a dose-dependent manner.

Mice were further evaluated for total body weight, liver, spleen andkidney weight. All changes were within the margin of error of theexperiment. No significant changes in body weight were observed duringthe treatment or at study termination. No significant changes in liveror spleen weights were observed.

The levels of ALT, AST, BUN, transaminases, plasma creatinine, glucoseand triglyceride were measured in mice treated with the short antisensecompounds. The levels of routine clinical indicators of liver and kidneyinjury and disease were within normal ranges and are not significantlychanged relative to saline-treated animals, demonstrating that the shortantisense compounds do not significantly affect renal or hepaticfunction. Triglyceride and glucose levels were not significantlyelevated relative to saline-treated animals.

Evaluation of ISIS 3796921-10-1 MOE Gapmer, ISIS 3921701-10-1Methyleneoxy BNA Gapmer, ISIS 3886252-8-2 MOE Gapmer and ISIS3921732-8-2 Methyleneoxy BNA Gapmer in Mice

The effect of ISIS 3796921-10-1 MOE gapmer and ISIS 3886252-8-2 MOEgapmer are compared with the effect of ISIS 3921701-10-1 MethyleneoxyBNA Gapmer and ISIS 3921732-8-2 Methyleneoxy BNA Gapmer (see Table 69)on mouse SGLT2 mRNA levels in vivo. Data are taken from threeexperiments in which male 6-week old Balb/c mice were dosed twice perweek for three weeks with 5, 25 and 125 nmol/kg of either the ISIS3796921-10-1 MOE gapmer or the ISIS 3886252-8-2 MOE gapmer given byintraperitoneal injection. Mice were sacrificed 48 hours following lastadministration and evaluated for SGLT2 mRNA levels in kidney. Targetlevels were determined by RT, real-time PCR as described by otherexamples herein. PCR results were normalized to an internal ISIScontrol. The data are expressed as percent change (“+” indicates anincrease, “−” indicates a decrease) relative to saline treated animalsand are illustrated in Table 69.

TABLE 69 Antisense inhibition of SGLT2 mRNA in vivo by a 1-10-1 and a2-8-2 MOE gapmer ISIS ISIS ISIS 392170 392173 Dose of 379692 1-10-1 ISIS2-8-2 oligonucleotide 1-10-1 Methyleneoxy 388625 Methyleneoxy nmol/kgMOE BNA 2-8-2 MOE BNA 125 −58 −69 −70 −75 25 −46 −54 −47 −57 5 −7 −23−18 −44

These results illustrate that both the 1-10-1 and 2-8-2 MOE gapmerinhibit the expression of SGLT2 in vivo at the highest three dosingranges in a dose-dependent manner. The results also illustrate that theMethyleneoxy BNA constructs are more potent then the MOE constructs. Nosignificant changes in body weight were observed during the treatment orat study termination. No significant changes in liver or spleen weightswere observed. The toxicity parameters including levels of ALT, AST,BUN, and creatinine were within normal ranges and are not significantlychanged relative to saline-treated animals, demonstrating that thecompounds do not significantly affect renal or hepatic function.

Evaluation of ISIS 3796921-10-1 MOE Gapmer and ISIS 3886252-8-2 MOEGapmer in Rat

The effect of ISIS 3796921-10-1 MOE gapmer and ISIS 388625 MOE 2-8-2gapmer (see Table 70) on rat SGLT2 mRNA levels in vivo. Data are takenfrom four experiments in which male Sprague-Dawley rats (170-200 g) weredosed twice per week for three weeks with 200, 50, 12.5, or 3.125nmol/kg of either the ISIS 3796921-10-1 MOE gapmer or the ISIS 3886252-8-2 MOE gapmer given by intraperitoneal injection. Rats weresacrificed 48 hours following last administration and evaluated forSGLT2 levels in kidney. Target levels were determined by RT, real-timePCR as described by other examples herein. PCR results were normalizedto an internal ISIS control. The results are shown below in Table 70.

TABLE 70 Antisense inhibition of SGLT2 mRNA in vivo by a 1-10-1 and a2-8-2 MOE gapmer % change in SGLT2 expression relative to saline Dose ofoligonucleotide ISIS 379692 ISIS 388625 umol/kg 1-10-1 2-8-2 200 −80 −8050 −65 −65 12.5 −15 −15 3.125 +30 +25

These results illustrate that both the 1-10-1 and 2-8-2 MOE gapmerinhibit the expression of SGLT2 in vivo at the highest three dosingranges in a dose-dependent manner.

Rats were further evaluated for total body weight, liver, spleen andkidney weight. All changes were within the margin of error of theexperiment. No significant changes in body weight were observed duringthe treatment or at study termination. No significant changes in liveror spleen weights were observed.

The levels of ALT, AST, BUN, cholesterol, plasma creatinine andtriglycerides were measured in rats treated with the short antisensecompounds. The levels of routine clinical indicators of liver and kidneyinjury and disease were within normal ranges and are not significantlychanged relative to saline-treated animals, demonstrating that the shortantisense compounds do not significantly affect renal or hepaticfunction.

Example 16 Antisense Inhibition of SGLT2 Expression in ZDF Rat

ISIS 388625, 388626 and control oligo ISIS 388628 were analyzed fortheir effect on ZDF rat plasma glucose levels and HbA1c. The leptinreceptor deficient Zucker diabetic fatty (ZDF) rat is a useful model forthe investigation of type 2 diabetes. Diabetes develops spontaneously inthese male rats at ages 8-10 weeks, and is associated with hyperphagia,polyuria, polydipsia, and impaired weight gain, symptoms which parallelthe clinical symptoms of diabetes (Phillips M S, et al., 1996, NatGenet. 13, 18-19). Six week old ZDF rats were injected intraperitoneallywith short antisense compound at a dose of 400 nM/kg once a week fortwelve weeks. Data are illustrated in Tables 71 and 72.

TABLE 71 Plasma glucose Plasma glucose levels Seq recorded on specificISIS ID dates (mg/dl) NO. NO Sequence (5′-3′) Motif Day 10 Day 40 Day 55Day 66 PBS n/a n/a 450.7 478.5 392.8 526.2 388625 246 TGTTCCAGCCCA 2-8-2MOE 435.5 278.7 213.8 325.5 388626 281 GGCATGAGCTTC 2-8-2 MOE 434.7300.5 219.8 379.8 388628 226 TAGCCGCCCACA 2-8-2 MOE 436.0 502.0 411.2668.8

TABLE 72 HbA1c Status Percentage HbA1c on specific dates (%) Seq p< 0.001 ISIS ID Day Day NO. NO Sequence (5′-3′) Motif 40 55 Day 68 PBSn/a n/a 8.0 8.9 10.0 388625 246 TGTTCCAGCCCA 2-8-2 MOE 6.5 5.8 4.3388626 281 GGCATGAGCTTC 2-8-2 MOE 6.6 5.9 4.0 388628 226 TAGCCGCCCACA2-8-2 MOE 8.0 9.1 7.8

ISIS 388625 and 388626 significantly reduced plasma glucose levels andHbA1C compared to PBS and control treated animals.

Example 17 Antisense Inhibition of SGLT2 Expression in Dog Kidney (ISIS388625)

ISIS 388625 is a 2-8-2 MOE Gapmer with sequence TGTTCCAGCCCA (SEQ ID NO:246) (e.g. see Table 71). The effect of ISIS 388625 on dog SGLT2 mRNAlevels. Data are taken from two dosing groups in which a total of ninemale beagle dogs were dosed with either one or ten mg/kg/week of ISIS388625 or saline given by subcutaneous injection twice weekly. On day 46of the study all dogs were sacrificed and evaluated for SGLT2 levels inkidney. Target levels were determined by quantitative RT, real-time PCRas described by other examples herein. PCR results were normalized to aninternal ISIS control. The results are shown below in Table 73.

TABLE 73 Antisense inhibition of SGLT2 mRNA in vivo by ISIS 388625 %change in SGLT2 expression Dose of oligonucleotide Relative to salinemg/kg/wk ISIS 388625 1 −85 10 −95

These results illustrate that greater than 80% reduction of SGLT2 mRNAcan be achieved at a 1 mg/kg/wk dose of ISIS 388625. Even greaterreduction can be achieved at slightly higher doses. Administration ofISIS 388625 in dog was also shown to improve glucose tolerance. Peakplasma glucose levels were decreased by over 50% on average and thesubsequent drop in glucose was lessened compared to saline controls in astandard glucose tolerance test. Urinary glucose excretion was alsoincreased.

Example 18 In Vivo Testing of Short Antisense Compounds Targeted toSGLT2 Nucleic Acid

Twenty 1-10-1 MOE gapmers that are complementary tohuman/monkey/mouse/rat SGLT2 were designed, synthesized and tested invivo for suppression of SGLT2 mRNA levels in kidney. Target sites formouse and rat are indicated in Table 74. Target sites for human areindicated in Tables 4 and 5. Data are averages from two experiments inwhich male 6-week old Balb/c mice were administered intraperitonealinjections of 350 nmol/kg of oligonucleotide, twice per week, over aperiod of two weeks (a total of four injections). Mice were sacrificed48 hours following the last administration and evaluated for SGLT2 mRNAlevels in kidney. SGLT2 mRNA levels were determined by quantitativereal-time PCR analysis according to standard procedures, using twodifferent PCR primer probe sets, primer probe set (PPS) 534 and PPS 553.SGLT2 mRNA levels were normalized to cyclophilin mRNA levels, which werealso measured by quantitative real-time PCR. The results are shown belowin Table 74.

TABLE 74 Antisense inhibition of SGLT2 in vivo 5′ Target 5′ Target Siteon Site on SEQ ID SEQ ID PPS PPS SEQ ISIS NO: XXX NO: XXX 534 % 553 % IDNO (mouse) (rat) Sequence (5′-3′) Motif Saline Saline NO PBS N/A — —370717 2684 152 TAGCCACCAACT 1-10-1 −84.4 −84.3 1554 MOE 379684 2070 64TGTCAGCAGGAT 1-10-1 −45.0 −43.2 214 MOE 379685 2103 97 TGACCAGCAGGA1-10-1 −10.3 −20.5 219 MOE 379686  2121* 115 ACCACAAGCCAA 1-10-1 −71.9−75.1 225 MOE 379687 2824 216 GATGTTGCTGGC 1-10-1 −47.1 −52.1 230 MOE379688 2876 268 CCAAGCCACTTG 1-10-1 −62.6 −70.4 240 MOE 379689 298AGAGCGCATTCC 1-10-1 −17.5 −30.4 241 MOE 379690 415 ACAGGTAGAGGC 1-10-1−18.9 −22.5 242 MOE 379691 454 AGATCTTGGTGA 1-10-1 −35.0 −48.6 243 MOE379692 508 TGTTCCAGCCCA 1-10-1 −88.1 −88.5 246 MOE 379693 546CATGGTGATGCC 1-10-1 −51.6 −59.9 254 MOE 379694 609 GACGAAGGTCTG 1-10-1−42.1 −54.4 264 MOE 379695 717 GGACACCGTCAG 1-10-1 −52.5 −64.1 266 MOE379696 954 CAGCTTCAGGTA 1-10-1 −24.6 −36.2 267 MOE 379697 982CTGGCATGACCA 1-10-1 −32.0 −46.3 272 MOE 379698 1071 GCAGCCCACCTC 1-10-1−11.8 −27.0 275 MOE 379699 1112 GGCATGAGCTTC 1-10-1 −83.5 −85.8 281 MOE379700 1138 CCAGCATGAGTC 1-10-1 −2.8 −16.4 285 MOE 379701 1210CCATGGTGAAGA 1-10-1 −0.3 −11.9 288 MOE 379702 1525 GCACACAGCTGC 1-10-1−87.8 −89.5 293 MOE 379703 1681 GCCGGAGACTGA 1-10-1 −44.2 −45.9 295 MOE*indicates 1 or 2 mismatches to a target sequence

Example 19 Antisense Inhibition of Human PCSK9 in Hep3B Cells

Short antisense compounds targeted to a PCSK9 nucleic acid were testedfor their effects on PCSK9 mRNA in vitro. The short antisense compoundsare presented in Table 6. The Isis No, gapmer motif and SEQ ID NO ofeach short antisense compound are shown again in Table 75. CulturedHep3B cells were treated with 100 nM of short antisense compound. 5-10-5MOE gapmers targeted to a PCSK9 nucleic acid were used as positivecontrols. After the treatment period, RNA was isolated from the cellsand PCSK9 mRNA levels were measured by quantitative real-time PCR, asdescribed herein. PCSK9 mRNA levels were adjusted according to total RNAcontent as measured by RIBOGREEN®. Results are presented in Table 75 aspercent inhibition of PCSK9 (% Inhib), relative to untreated controlcells. In the “% Inhib” column, a “0” indicates that no reduction ofPCSK9 mRNA was observed with that particular short antisense compound.

TABLE 75 Antisense inhibition of PCSK9 by short antisense compounds 5′3′ Target Target Site on Site on % ISIS SEQ ID SEQ ID SEQ ID GapmerInhibition No. NO NO: 4 NO: 4 Motif Range % Inhib 400297 329 695 7082-10-2 MOE 0 400298 330 696 709 2-10-2 MOE 0 400299 331 697 710 2-10-2MOE 0 400300 332 742 755 2-10-2 MOE 9 400301 333 757 770 2-10-2 MOE20-30% 27 400302 334 828 841 2-10-2 MOE 0 400303 335 829 842 2-10-2 MOE0 400304 336 830 843 2-10-2 MOE 10-20% 11 400305 337 937 950 2-10-2 MOE30-40% 38 400306 338 952 965 2-10-2 MOE 40-50% 40 400307 339 988 10012-10-2 MOE 70-80% 76 400308 340 989 1002 2-10-2 MOE 50-60% 55 400309 341990 1003 2-10-2 MOE 40-50% 44 400310 342 991 1004 2-10-2 MOE 8 400311343 992 1005 2-10-2 MOE 10-20% 18 400312 344 993 1006 2-10-2 MOE 20-30%28 400313 345 994 1007 2-10-2 MOE 10-20% 10 400314 346 1057 1070 2-10-2MOE 20-30% 26 400315 347 1075 1088 2-10-2 MOE 0 400316 348 1076 10892-10-2 MOE 8 400317 349 1077 1090 2-10-2 MOE 7 400318 350 1078 10912-10-2 MOE 20-30% 26 400319 351 1093 1106 2-10-2 MOE 0 400320 352 10941107 2-10-2 MOE 0 400321 353 1095 1108 2-10-2 MOE 0 400322 354 1096 11092-10-2 MOE 0 400323 355 1147 1160 2-10-2 MOE 0 400324 356 1255 12682-10-2 MOE 7 400325 357 1334 1347 2-10-2 MOE 4 400326 358 1335 13482-10-2 MOE 0 400327 359 1336 1349 2-10-2 MOE 30-40% 36 400328 360 14531466 2-10-2 MOE 10-20% 13 400329 361 1454 1467 2-10-2 MOE 10-20% 14400330 362 1455 1468 2-10-2 MOE 40-50% 43 400331 363 1456 1469 2-10-2MOE 30-40% 35 400332 364 1569 1582 2-10-2 MOE 0 400333 365 1570 15832-10-2 MOE 0 400334 366 1571 1584 2-10-2 MOE 0 400335 367 1572 15852-10-2 MOE 0 400336 368 1573 1586 2-10-2 MOE 4 400337 369 1574 15872-10-2 MOE 0 400338 370 1575 1588 2-10-2 MOE 9 400339 371 1576 15892-10-2 MOE 0 400340 372 1577 1590 2-10-2 MOE 0 400341 373 1578 15912-10-2 MOE 0 400342 374 1621 1634 2-10-2 MOE 0 400343 375 1622 16352-10-2 MOE 0 400344 376 1623 1636 2-10-2 MOE 0 400345 377 1624 16372-10-2 MOE 0 400346 378 1738 1751 2-10-2 MOE 5 400347 379 1739 17522-10-2 MOE 0 400348 380 1740 1753 2-10-2 MOE 0 400349 381 1741 17542-10-2 MOE 10-20% 13 400350 382 1834 1847 2-10-2 MOE 10-20% 15 400351383 1835 1848 2-10-2 MOE 10-20% 14 400352 384 1836 1849 2-10-2 MOE20-30% 29 400353 385 1837 1850 2-10-2 MOE 10-20% 19 400354 386 1838 18512-10-2 MOE 10-20% 19 400355 387 1839 1852 2-10-2 MOE 0 400356 388 18401853 2-10-2 MOE 0 400357 389 2083 2096 2-10-2 MOE 0 400358 390 2084 20972-10-2 MOE 10-20% 12 400359 391 2085 2098 2-10-2 MOE 0 400360 392 20862099 2-10-2 MOE 30-40% 38 400361 393 2316 2329 2-10-2 MOE 2 400362 3942317 2330 2-10-2 MOE 10-20% 16 400363 395 2318 2331 2-10-2 MOE 8 400364396 2319 2332 2-10-2 MOE 0 400365 397 2320 2333 2-10-2 MOE 20-30% 25400366 398 2321 2334 2-10-2 MOE 10-20% 15 400367 399 2322 2335 2-10-2MOE 10-20% 12 400368 400 2323 2336 2-10-2 MOE 10-20% 11 400369 401 23242337 2-10-2 MOE 0 400370 402 2325 2338 2-10-2 MOE 10-20% 13 400371 4033543 3556 2-10-2 MOE 0

As illustrated in Table 75, short antisense compounds targeted to aPCSK9 nucleic acid, having a 2-10-2 MOE gapmer motif, reduced PCSK9 mRNAin cultured cells.

Short antisense compounds targeted to a PCSK9 nucleic acid were testedin a dose response experiment Hep3B cells. Cells were treated asdescribed herein with n-M concentrations of short antisense compound asindicated in Tables 76. After the treatment period, RNA was isolatedfrom the cells and PCSK9 mRNA levels were measured by quantitativereal-time PCR, as described herein. PCSK9 mRNA levels were normalized tocyclophilin mRNA levels, as measured by real-time PCR using acyclophilin-specific primer probe set. Results are presented as percentinhibition of PCSK9, relative to untreated control cells. Also shown isthe EC₅₀ (concentration at which 50% reduction of mRNA is observed) foreach short antisense compound tested in the dose response experiment, ascalculated using Graphpad Prism. As illustrated in the following table,PCSK9 mRNA levels were reduced in a dose-dependent manner.

TABLE 76 Dose-dependent antisense inhibition of PCSK9 by short antisensecompounds % Inhibition 160 nM 80 nM 40 nM 20 nM 10 nM 5 nM 5-10-5 95 9685 78 58 38 400307 93 92 56 45 39 35 400308 86 77 40 26 10 31 400309 7872 12 38 23 49 400327 55 43 49 23 37 5 400330 71 82 69 40 32 8 400331 8275 63 47 40 29 400352 64 63 44 40 16 7 400353 48 54 43 23 27 15

Example 20 Antisense Inhibition of PCSK9 by Short Antisense CompoundsComprising BNAs

Short antisense compounds targeted to a PCSK9 nucleic acid were testedin dose response experiments, in both mouse and human cultured cells.The compounds tested included ISIS 403739 and ISIS 403740. ISIS 403739is a short antisense compound consisting of the nucleotide sequence ofSEQ ID NO: 404 and having a 2-10-2 gapmer motif, where the nucleotidesin the wings comprise (6′S)-6′methyl BNA. ISIS 403740 is a shortantisense compound consisting of the nucleotide sequence of SEQ ID NO:405 and having a 2-10-2 gapmer motif, where the nucleotides in the wingscomprise (6′S)-6′methyl BNA. Also tested was a 5-10-5 MOE gapmertargeted to a PCSK9 nucleic acid.

Mouse hepatocytes were plated and treated as described herein with nMconcentrations of short antisense compound as indicated in Table 77.After the treatment period, RNA was isolated from the cells and PCSK9mRNA levels were measured by quantitative real-time PCR, as describedherein. PCSK9 mRNA levels were normalized to cyclophilin mRNA levels, asmeasured by real-time PCR using a cyclophilin-specific primer probe set.Results are presented as percent inhibition of PCSK9, relative tountreated control cells. Where present, “0” indicates no observedreduction in PCSK9 mRNA. ISIS 403739 exhibited dose-dependent reductionof mouse PCSK9 mRNA at the doses of 30 nM and higher. ISIS 403740exhibited reduction of mouse PCSK9 mRNA at the two highest doses ofshort antisense compound.

TABLE 77 Antisense inhibition of mouse PCSK9 by short antisensecompounds comprising BNAs % Inhibition 3.75 nM 7.5 nM 15 nM 30 nM 60 nM120 nM 240 nM 5-10-5 10 15 21 18 44 43 77 403739 40 19 29 29 32 49 57403740 3 0 29 13 0 40 33

Human Hep3B cells were treated with nM concentrations of short antisensecompound as described herein. After the treatment period, RNA wasisolated from the cells and PCSK9 mRNA levels were measured byquantitative real-time PCR, as described herein. PCSK9 mRNA levels werenormalized to cyclophilin mRNA levels, as measured by real-time PCRusing a cyclophilin-specific primer probe set. Results are presented aspercent inhibition of PCSK9, relative to untreated control cells. Thedata are shown in Table 78 and demonstrate a dose-dependent reduction inhuman PCSK9 mRNA following treatment with ISIS 403740. ISIS 403739exhibited dose-dependent reduction at higher doses.

TABLE 78 Antisense inhibition of mouse PCSK9 by short antisensecompounds comprising BNAs % Inhibition 2.5 nM 5 nM 10 nM 20 nM 40 nM 80nM 160 nM 5-10-5 7 2 21 33 30 59 71 403739 10 5 7 6 25 52 65 403740 6 1216 29 45 48 59

Example 21 Antisense Inhibition of GCGR in HepG2 Cells

Short antisense compounds targeted to a GCGR nucleic acid were testedfor their effects on GCGR mRNA in vitro.

HepG2 Cells

Cultured HepG2 cells at a density of 10000 cells per well in a 96-wellplate were treated as described herein with 25, 50, 100 or 200 nM ofantisense oligonucleotide. After the treatment period, RNA was isolatedfrom the cells and GCGR mRNA levels were measured by quantitativereal-time PCR, as described herein. GCGR mRNA levels were adjustedaccording to total RNA content as measured by RIBOGREEN®. Results arepresented as percent reduction in GCGR mRNA, relative to untreatedcontrol cells.

Table 79 presents data following treatment with the indicated doses ofISIS 327161, a 3-10-3 MOE gapmer. ISIS 327161 reduced GCGR mRNA in adose-dependent manner.

TABLE 79 Antisense inhibition of GCGR in HepG2 cells by a shortantisense compound ISIS Seq ID Gapmer 25 50 100 200 NO. NO Sequence(5′-3′) Motif nM nM nM nM 327161 520 AGCTGCTGTACATC 3-8-3 −36 −30 −33−64 MOE

Monkey Hepatocytes

Additional short antisense compounds targeted to a GCGR nucleic acidwere tested for their effects on monkey GCGR mRNA in vitro. Culturedprimary monkey hepatocytes were treated as described herein with 25, 50,100 or 200 nM of short antisense compound. After the treatment period,RNA was isolated from the cells and GCGR mRNA levels were measured byquantitative real-time PCR, as described herein. GCGR mRNA levels wereadjusted according to total RNA content as measured by RIBOGREEN®.Results are presented in Table 80 as percent reduction in GCGR mRNA,relative to untreated control cells.

TABLE 80 Antisense inhibition of GCGR in primary monkey hepatocytes byshort antisense compounds ISIS Seq ID Gapmer 25 50 100 200 NO. NOSequence (5′-3′) Motif nM nM nM nM 327131 489 ATGTTGGCCGTGGT 3-8-3 0 −8−36 −36 MOE 327161 520 AGCTGCTGTACATC 3-8-3 −19 −33 −55 −54 MOE

Example 22 Antisense Inhibition of DGAT2 by Short Antisense Compounds

Short antisense compounds targeted to a DGAT2 nucleic acid were testedfor their effects on DGAT2 mRNA in vitro. Cultured A10 cells in a96-well plate were treated with 75 nM of short antisense compound. Aftera treatment period of approximately 24 hours, RNA was isolated from thecells and DGAT2 mRNA levels were measured by quantitative real-time PCR,as described herein. DGAT2 mRNA levels were adjusted according to totalRNA content as measured by RIBOGREEN®. Results are presented as percentinhibition of DGAT2, relative to untreated control cells in Table 81.

TABLE 81 Antisense inhibition of DGAT2 in A10 cells ISIS Seq ID GapmerNO. NO Sequence (5′-3′) Motif % Control 372491 795 ACATGAGGATGACACT3-10-3 MOE 80 372500 702 GTGTGTCTTCACCAGC 3-10-3 MOE 16 372501 704TTGTGTGTCTTCACCA 3-10-3 MOE 28 372503 708 GCAGGTTGTGTGTCTT 3-10-3 MOE 35372508 719 AGTTCCTGGTGGTCAG 3-10-3 MOE 35 372516 805 TACAGAAGGCACCCAG3-10-3 MOE 27 372524 738 GCCAGGCATGGAGCTC 3-10-3 MOE 21 372530 746TCGGCCCCAGGAGCCC 3-10-3 MOE 35 372546 825 TTGGTCTTGTGATTGT 3-10-3 MOE 34372563 691 AGCCAGGTGACAGA 2-10-2 MOE 48 372569 796 CATGAGGATGACAC 2-10-2MOE 104 372578 703 TGTGTCTTCACCAG 2-10-2 MOE 59 372580 707GGTTGTGTGTCTTC 2-10-2 MOE 48 372586 720 GTTCCTGGTGGTCA 2-10-2 MOE 40372594 806 ACAGAAGGCACCCA 2-10-2 MOE 77 372602 739 CCAGGCATGGAGCT 2-10-2MOE 39 372618 765 GTGGTACAGGTCGA 2-10-2 MOE 29 372624 826 TGGTCTTGTGATTG2-10-2 MOE 56

Additional short antisense compounds targeted to DGAT2 mRNA were testedin vitro in a dose-response experiment. A10 cells were prepared asdescribed above and treated with 6.25, 12.5, 25.0, 50.0, 100.0, and200.0 nM short antisense compounds to determine if DGAT2 inhibitionoccurs in a dose-dependent manner. The data demonstrate that each of theshort antisense compounds presented in Table 82 reduces rat DGAT2 mRNAin a dose-dependent manner. Results are presented as percent inhibition,relative to untreated control cells. A “0” indicates that DGAT2 mRNA wasnot reduced.

TABLE 82 Dose-Dependent Inhibition of DGAT2 in A10 cells Seq ISIS IDGapmer NO. NO Sequence (5′-3′) Motif 6.25 nM 12.5 nM 25.0 nM 50.0 nM100.0 nM 200.0 nM 372562 784 GTCTTGGAGGGCCG 2-10-2 0 0 0 36 48 75 MOE372568 794 GACACTGCAGGCCA 2-10-2 0 0 15 26 72 69 MOE 372586 720GTTCCTGGTGGTCA 2-10-2 19 0 7 22 45 77 MOE 372602 739 CCAGGCATGGAGCT2-10-2 0 0 0 18 47 76 MOE 372618 765 GTGGTACAGGTCGA 2-10-2 0 5 0 27 6580 MOE

Additional short antisense compounds targeted to DGAT2 mRNA were testedin vitro. A10 cells were prepared as described above and treated with0.62, 1.85, 5.56, 16.67, 50.0, and 150.0 nM short antisense compounds todetermine if DGAT2 inhibition occurs in a dose-dependent manner. DGAT2mRNA was measured using quantitative real-time PCR, as described herein.The data demonstrate that each of the short antisense compoundspresented in Table 83 below inhibit rat DGAT2 mRNA in a dose-dependentmanner. Results are presented as percent inhibition of rat DGAT2,relative to untreated control cells. Where present, “0” indicates thatno reduction in DGAT2 mRNA was observed.

TABLE 83 Dose-Dependent Inhibition of DGAT2 in A10 cells Seq ISIS IDGapmer NO. NO Sequence (5′-3′) Motif 0.62 nM 1.85 nM 5.56 nM 16.67 nM 50nM 150 nM 372500 702 GTGTGTCTTCACCAGC 3-10-3 0 0 0 18 64 88 MOE 372501704 TTGTGTGTCTTCACCA 3-10-3 1 5 10 11 25 68 MOE 372503 708GCAGGTTGTGTGTCTT 3-10-3 7 10 4 25 54 80 MOE 372508 719 AGTTCCTGGTGGTCAG3-10-3 0 0 6 14 39 71 MOE 372516 805 TACAGAAGGCACCCAG 3-10-3 1 10 0 4 3581 MOE 372524 738 GCCAGGCATGGAGCTC 3-10-3 7 0 5 30 68 91 MOE 372530 746TCGGCCCCAGGAGCCC 3-10-3 0 2 0 10 38 78 MOE 372546 825 TTGGTCTTGTGATTGT3-10-3 0 2 11 4 48 78 MOE 372563 691 AGCCAGGTGACAGA 2-10-2 0 0 0 1 4 46MOE 372578 703 TGTGTCTTCACCAG 2-10-2 0 0 0 2 7 42 MOE 372580 707GGTTGTGTGTCTTC 2-10-2 0 5 5 3 16 42 MOE 372586 720 GTTCCTGGTGGTCA 2-10-20 0 0 0 7 55 MOE 372594 806 ACAGAAGGCACCCA 2-10-2 0 0 0 0 2 15 MOE372602 739 CCAGGCATGGAGCT 2-10-2 0 0 10 0 19 51 MOE 372618 765GTGGTACAGGTCGA 2-10-2 0 0 0 0 30 60 MOE 372624 826 TGGTCTTGTGATTG 2-10-20 0 0 1 16 38 MOE

Example 23 Antisense Inhibition of Human PTP1B in HuVEC Cells

Short antisense compounds targeted to a PTP1B nucleic acid were testedfor their effects on PTP1B mRNA in vitro. Cultured HuVEC cells at adensity of 5000 cells per well in a 96-well plate were treated asdescribed herein with 3 nM of short antisense compound. After thetreatment period, RNA was isolated from the cells and PTP1B mRNA levelswere measured by quantitative real-time PCR, as described herein. PTP1BmRNA levels were adjusted according to total RNA content as measured byRIBOGREEN®. Results are presented as percent inhibition of PTP1B (%Inhib), relative to untreated control cells. The data demonstrated thatshort antisense compounds targeted to a PTP1B nucleic acid and having a2-10-2 gapmer motif can inhibit PTP1B in HuVEC cells in Table 84.

TABLE 84 Antisense inhibition of PTP1B in HuVEC cells by short antisensecompounds ISIS NO. SEQ ID NO Gapmer Motif % Inhib 399301 1542 2-10-2 OMe55 404137 1053 2-10-2 MOE 76 404138 1054 2-10-2 MOE 76 404139 10522-10-2 MOE 80 404140 1051 2-10-2 MOE 73

Example 24 Antisense Inhibition of Human PTP1B in HepG2 Cells

Short antisense compounds targeted to a PTP1B nucleic acid were testedfor their effects on PTP1B mRNA in vitro. Cultured HepG2 cells at adensity of 10000 cells per well in a 96-well plate were treated with 25nM of antisense oligonucleotide. After the treatment period, RNA wasisolated from the cells and PTP1B mRNA levels were measured byquantitative real-time PCR, as described herein. PTP1B mRNA levels wereadjusted according to total RNA content as measured by RIBOGREEN®.Results are presented as percent inhibition (% Ihib) of PTP1B, relativeto untreated control cells. The data demonstrated that short antisensecompounds targeted to a PTP1B nucleic acid and having a 2-10-2 gapmermotif can inhibit PTP1B in HepG2 cells in Table 85.

TABLE 85 Antisense inhibition of PTP1B in HepG2 cells by short antisensecompounds ISIS NO. SEQ ID NO Gapmer Motif % Inhib 399301 1542 2-10-2 OMe43 404137 1053 2-10-2 MOE 71 404138 1054 2-10-2 MOE 86 404139 10522-10-2 MOE 45 404140 1051 2-10-2 MOE 93

Example 25 Antisense Inhibition of PTP1B in HuVEC Cells: Dose ResponseExperiment

Human vascular endothelial (HuVEC) cells were plated at a density of5000 cells per well and treated as described herein with nMconcentrations of short antisense compound as indicated in Table 86.After the treatment period, RNA was isolated from the cells and PTP1BmRNA levels were measured by quantitative real-time PCR, as describedherein. PTP1B mRNA levels were adjusted according to total RNA contentas measured by RIBOGREEN®. Two different human PTP1B primer probe setswere used to measure mRNA levels. Results with Primer Probe Set (PPS)198 are shown in Table 86, and results with Primer Probe Set (PPS) 3000are shown in Table 87. Results are presented as percent inhibition ofPTP1B mRNA expression relative to untreated control cells. Wherepresent, “0” indicates that no PTP1B mRNA reduction was observed. Asillustrated in Tables 86 and 87, PTP1B mRNA levels were reduced in adose-dependent manner.

TABLE 86 Dose Response for Human PTP1B in HuVEC cells, using PPS 198 %Inhibition Seq ID Gapmer 1.11 30.0 ISIS NO. NO Motif nM 3.33 nM 10.0 nMnM 398105 1066 2-10-2 MOE 0 25 79 90 398112 1072 2-10-2 MOE 1 10 73 93398120 1086 2-10-2 MOE 0 31 80 96 399096 1544 2-10-2 MOE 3 30 78 96399102 1545 2-10-2 MOE 0 15 62 88 399113 1547 2-10-2 MOE 0 31 72 90399132 1548 2-10-2 MOE 0 32 75 95 399173 1549 2-10-2 MOE 0 24 63 89399208 1550 2-10-2 MOE 0 37 86 93 399276 1551 2-10-2 MOE 0 8 61 89399301 1542 2-10-2 MOE 8 63 91 97 399315 1552 2-10-2 MOE 0 20 68 88398173 1543 1-10-1 MOE 0 4 80 97

TABLE 87 Dose Response for Human PTP1B in HuVEC cells, using PPS 3000 %Inhibition Seq ID Gapmer 1.11 30.0 ISIS NO. NO Motif nM 3.33 nM 10.0 nMnM 398105 1066 2-10-2 MOE 0 35 79 93 398112 1072 2-10-2 MOE 0 26 77 94398120 1086 2-10-2 MOE 0 35 79 93 399096 1544 2-10-2 MOE 0 23 75 94399102 1545 2-10-2 MOE 0 9 60 87 399113 1547 2-10-2 MOE 0 9 65 90 3991321548 2-10-2 MOE 0 26 76 91 399173 1549 2-10-2 MOE 0 11 59 92 399208 15502-10-2 MOE 0 47 85 96 399276 1551 2-10-2 MOE 0 14 64 86 399301 15422-10-2 MOE 16 65 93 99 399315 1552 2-10-2 MOE 0 25 71 93 398173 15431-10-1 MOE 0 18 80 90

Example 26 Antisense Inhibition of ApoB by Short Antisense Compounds

The short antisense compounds shown in Table 88 were tested for theireffects in vivo. Six-week old male Balb/c mice (Jackson Laboratory, BarHarbor, Me.) were administered intraperitoneal doses of 3.2, 1, 0.32, or0.1 umol/kg, twice per week for three weeks. A 5-10-5 MOE gapmer wasused for a control treatment. Mice were sacrificed approximately 48hours following the final dose. Liver tissue was collected for RNAisolation, and blood was collected for serum chemistry analyses. ApoBmRNA levels were measured by real-time PCR as described herein. ApoBmRNA levels were normalized to RNA levels as determined by RIBOGREEN,and are presented in Table 89 as percent inhibition relative to ApoBmRNA levels in saline-treated control animals.

TABLE 88 Short Antisense Compounds Targeting an ApoB nucleic acid SEQISIS Sequence ID NO (5′-3′) Gapmer Motif NO 387462 GGTACATGGAAGTC 2-10-2Methyleneoxy BNA 190 398296 GGTACATGGAAGTC 2-10-2 190 6′-(S)-methylMethyleneoxy BNA

TABLE 89 Antisense inhibition of ApoB by Short Antisense CompoundsComprising BNA Dose Isis No (umol/kg) % Inhib 379818 1 56 387462 0.1 330.32 57 1 93 3.2 99 398296 0.1 17 0.32 35 1 80 3.2 98

Table 89 shows that ApoB mRNA levels were reduced in a dose-dependentmanner following treatment with short antisense compounds having a2-10-2 gapmer motif and BNA modifications in the wings. At the 1 umol/kgdose, ApoB inhibition by the short antisense compounds was greater thanobserved with a 5-10-5 MOE gapmer at an equivalent dose. Cholesterol wasreduced at the 1 and 3.2 umol/kg doses of short antisense compound.

The short antisense compounds exhibited little to no adverse sideeffects, as judged by organ and body weights, serum transaminases,bilirubin, blood urea nitrogen, and creatinine.

Example 27 Antisense Inhibition of PTEN by Short Antisense Compounds

The short antisense compounds shown in Table 90 were tested for theireffects in vivo. Six-week old male Balb/c mice (Jackson Laboratory, BarHarbor, Me.) were administered intraperitoneal doses of 3.2, 1, 0.32, or0.1 umol/kg, twice per week for three weeks. A 5-10-5 MOE gapmer wasused for a control treatment. Mice were sacrificed approximately 48hours following the final dose. Liver tissue was collected for RNAisolation, and blood was collected for serum chemistry analyses. PTENmRNA levels were measured by real-time PCR as described herein. PTENmRNA levels were normalized to RNA levels as determined by RIBOGREEN,and are presented in Table 91 as percent inhibition relative to PTENmRNA levels in saline-treated control animals.

TABLE 90 Short Antisense Compounds targeted to a PTEN nucleic acid ISISSequence SEQ ID NO (5′-3′) Gapmer Motif NO 392063 AGGCCAGTGCTAAG 2-10-21226 Methyleneoxy BNA 392749 AGGCCAGTGCTAAG 2-10-2 (6′S)-6′-methyl 1226Methyleneoxy BNA 396006 AGGCCAGTGCTAAG 2-10-2 1226 alpha-L-methyleneoxyBNA

TABLE 91 Antisense inhibition of PTEN by short antisense compoundscomprising BNA modifications Dose Isis No (umol/kg) % Inhib 116847 1 47392063 0.1 26 0.32 43 1 74 3.2 96 392749 0.1 17 0.32 34 1 64 3.2 96396006 0.1 20 0.32 32 1 67 3.2 88

Table 91 shows that PTEN mRNA levels were reduced in a dose-dependentmanner following treatment with short antisense compounds having a2-10-2 gapmer motif and BNA modifications in the wings. At the 1 umol/kgdose, PTEN inhibition by the short antisense compounds was greater thanobserved with a 5-10-5 MOE gapmer at an equivalent dose.

With the exception of the highest dose of ISIS 392063, no significantincreases in serum transaminases were observed. Overall, the shortantisense compounds exhibited little to no adverse side effects.

Example 28 Single Dose Administration of Short Antisense CompoundsComprising BNA Modifications

Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereadministered a single intraperitoneal injection of short antisensecompound at a dose of 8, 4, 2 or 1 μmol/kg. The short antisensecompounds tested were ISIS 387462 and ISIS 398296. Each dose groupconsisted of four animals. A 5-10-5 MOE gapmer was used for a controltreatment. Mice were sacrificed approximately 48 hours following thefinal dose. Liver tissue was collected for RNA isolation, and blood wascollected for serum chemistry analyses. ApoB mRNA levels were measuredby real-time PCR as described herein. ApoB mRNA levels were normalizedto RNA levels as determined by RIBOGREEN, and are presented in Table 92as percent inhibition relative to ApoB mRNA levels in saline-treatedcontrol animals.

TABLE 92 Antisense inhibition of ApoB by Short Antisense CompoundsComprising BNA Dose Isis No (umol/kg) % Inhib 379818 8 77 387462 8 99 493 2 81 1 58 398296 8 97 4 81 2 54 1 19

Table 92 shows that ApoB mRNA levels were reduced in a dose-dependentmanner following a single administration of short antisense compoundshaving a 2-10-2 gapmer motif and BNA modifications in the wings. At the8 umol/kg dose, ApoB inhibition by the short antisense compounds wasgreater than observed with a 5-10-5 MOE gapmer at an equivalent dose.The ED₅₀ of ISIS 387462 was 3.9 mg/kg, and the ED₅₀ of ISIS 398296 was8.7 mg/kg. Cholesterol was also reduced in a dose-dependent manner.Triglycerides were reduced at the highest dose.

The short antisense compounds exhibited little to no adverse sideeffects, as judged by organ and body weights, serum transaminases,bilirubin, blood urea nitrogen, and creatinine.

In a similar single dose administration study, ISIS 392748, having SEQID NO: 1226, a 2-10-2 gapmer motif, where the nucleotides of the wingscomprise (6′R)-6′-methyl methyleneoxy BNA modifications, reduced PTENmRNA in a dose-dependent manner. Additionally, ISIS 392749, having SEQID NO: 1226, a 2-10-2 gapmer motif, where the nucleotides of the wingscomprise (6′S)-6′-methyl methyleneoxy BNA modifications, reduced PTENmRNA in a dose-dependent manner. A short antisense compound having2-10-2 gapmer motifs, the sequence of SEQ ID NO: 1226, and6-(S)—CH2-O—CH3-BNA modifications also reduced PTEN mRNA in a similar invivo study. A short antisense compound having 2-10-2 gapmer motifs, thesequence of SEQ ID NO: 1226, and 6-(R)—CH2-O—CH3-BNA modifications alsoreduced PTEN mRNA in a similar in vivo study.

Example 29 Single Dose Administration of Short Antisense CompoundsComprising BNA Modifications

Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereadministered a single intraperitoneal injection of antisense compound ata dose of 8, 4, 2 or 1 μmol/kg. Each dose group consisted of fouranimals. The compounds tested were ISIS 392063, ISIS 392749, and ISIS366006. A 5-10-5 MOE gapmer was used for a control treatment. Mice weresacrificed approximately 48 hours following the final dose. Liver tissuewas collected for RNA isolation, and blood was collected for serumchemistry analyses. ApoB mRNA levels were measured by real-time PCR asdescribed herein. ApoB mRNA levels were normalized to RNA levels asdetermined by RIBOGREEN, and are presented in Table 93 as percentinhibition relative to ApoB mRNA levels in saline-treated controlanimals.

TABLE 93 Antisense inhibition of PTEN by short antisense compoundscomprising BNA modifications Dose Isis No (umol/kg) % Inhib 116847 8 62392063 8 92 4 82 2 58 1 38 396565 8 76 4 38 2 24 1 11 396006 8 94 4 82 248 1 18

Table 93 shows that PTEN mRNA levels were reduced in a dose-dependentmanner following treatment with short antisense compounds having a2-10-2 gapmer motif and BNA modifications in the wings. At the 8 umol/kgdose, PTEN inhibition by the short antisense compounds was greater thanobserved with a 5-10-5 MOE gapmer at an equivalent dose. The estimatedED₅₀s were 7 mg/kg for ISIS 392063, 17.4 mg/kg for ISIS 396565, and 9.3mg/kg for ISIS 396006.

With the exception of the highest dose of ISIS 392063, no significantincreases in serum transaminases were observed. Overall, the shortantisense compounds exhibited little to no adverse side effects.

Example 30 Antisense Inhibition of ApoB by Short Antisense CompoundsComprising Palmitic Acid Conjugates

Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereadministered a single intraperitoneal injection of antisense compound ata dose of 2.5, 1.0. 0.4, and 0.16 umol/kg. Each dose group consisted offour animals. The compounds tested are shown in Table 94. A 5-10-5 MOEgapmer was used for a control treatment. Mice were sacrificedapproximately 48 hours following the final dose. Liver tissue wascollected for RNA isolation, and blood was collected for serum chemistryanalyses. ApoB mRNA levels were measured by real-time PCR as describedherein. ApoB mRNA levels were normalized to RNA levels as determined byRIBOGREEN, and are presented in Table 95 as percent inhibition relativeto ApoB mRNA levels in saline-treated control animals.

TABLE 94 Short antisense compounds comprising palmitic conjugates ISISSEQ NO Sequence (5′-3′) Gapmer Motif ID NO 387462 GGTACATGGAAGTC 2-10-2Methyleneoxy BNA 190 391871 GGTACATGGAAGTC 1-1-10-2 2′-(butylacetomido)-190 palmitamide/MOE/MOE Unmodified cytosines in gap (i.e., 2-10-2 MOEwith 2′- (butylacetomido)-palmitamide substituted at 5′ nucleotide391872 GGTACATGGAAGTC 1-1-10-2 2′-(butylacetomido)- 190 palmitamideMethyleneoxy BNA/Methyleneoxy BNA Unmodified cytosines in gap (i.e.,2-10-2 methyleneoxy BNA with 2′-(butylacetomido)- palmitamidesubstituted at 5′ nucleotide)

TABLE 95 Antisense inhibition by short antisense compounds comprisingpalmitic acid conjugates Dose Isis No (umol/kg) % Inhib 5-10-5 2.5 54387462 2.5 99 1.0 91 0.4 65 0.16 16 391871 2.5 49 1.0 18 0.4 5 0.16 0391872 2.5 99 1.0 92 0.4 50 0.16 18

Table 95 shows that ApoB mRNA levels were reduced in a dose-dependentmanner following treatment with short antisense compounds having apalmitic acid (C16) conjugate. At the 2.5 umol/kg dose, ApoB inhibitionby the short antisense compounds was greater than observed with a 5-10-5MOE gapmer at an equivalent dose. In this study, the estimated ED₅₀swere 1.5 mg/kg for ISIS 387462, 13.1 mg/kg for ISIS 391871, and 1.9mg/kg for ISIS 391872. The estimated ED₅₀ for the 5-10-5 MOE gapmer was17.4 mg/kg. Triglycerides were reduced at the 2.5 and 1.0 mg/kg doses ofISIS 387462 and ISIS 391872. ISIS 387462 and ISIS 391872 markedlyreduced total cholesterol, HDL-C and LDL-C in a dose-dependent manner;reduction in LDL-C was so marked that it fell below the limit ofdetection. Overall, the short antisense compounds exhibited little to noadverse effects.

Example 31 Antisense Inhibition of PCSK9 In Vivo by Short AntisenseCompounds Comprising BNA Modifications

Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereadministered a single intraperitoneal injection of antisense compound ata dose of 15, 4.7, 1.5 and 0.47 umol/kg of ISIS 403739 or 403740. Eachdose group consisted of four animals. A 5-10-5 MOE gapmer was used for acontrol treatment. Mice were sacrificed approximately 72 hours followingthe final dose. Liver tissue was collected for RNA isolation, and bloodwas collected for serum chemistry analyses. PCSK9 mRNA levels weremeasured by real-time PCR as described herein. PCSK9 mRNA levels werenormalized to cyclophilin mRNA levels as determined by real-time PCR.ISIS 403739 reduced PCSK9 mRNA by approximately 70%, relative to salinecontrols. ISIS 403740 reduced PCSK9 by approximately 13% relative tosaline controls, however, the reduction was not statisticallysignificant. The lower doses did not significantly reduce PCSK9 mRNA.Overall, the short antisense compounds exhibited little to no adverseside effects.

1. A short antisense compound 8 to 16 monomers in length, comprising a2′-deoxyribonucleotide gap region flanked on each side by a wing,wherein each wing independently comprises 1 to 3 high-affinity modifiedmonomers and wherein the short antisense compound is targeted to anucleotide encoding GCCR.
 2. The short antisense compound of claim 1,wherein said high-affinity modified monomers are sugar-modifiednucleotides.
 3. The short antisense compound of claim 2, wherein atleast one of the sugar-modified nucleotides comprises a bridge betweenthe 4′ and the 2′ position of the sugar.
 4. The short antisense compoundof claim 2, wherein each of said high-affinity modified nucleotidesconfers a T_(m) of 1 to 4 degrees per nucleotide.
 5. The short antisensecompound of claim 2, wherein each of said sugar-modified nucleotidescomprises a 2′-substituent group that is other than H or OH.
 6. Theshort antisense compound of claim 5, wherein at least one of saidsugar-modified nucleotides is a 4′ to 2′ bridged bicyclic nucleotide. 7.The short antisense compound of claim 5, wherein each of the2′-substituent groups is, independently, alkoxy, substituted alkoxy, orhalogen.
 8. The short antisense compound of claim 7, wherein each of the2′-substituent groups is OCH₂CH₂OCH₃.
 9. The short antisense compoundclaim 3, wherein the conformation of each of said sugar-modifiednucleotides is, independently, β-D or α-L.
 10. The short antisensecompound claim 5, wherein each of said bridges independently comprises 1or from 2 to 4 linked groups independently selected from—[0C(R₁)(R₂)]_(n)—, —C(R₁)═C(R₂)—, —C(R₁)═N—, —C(═NR₁)—, —C(═O)—,—C(═S)—, —O—, —Si(R₁)₂—, —S(═O)_(n)— and —N(R₁)—; wherein x is 0, 1, or2; n is 1, 2, 3, or 4; each R₁ and R₂ is, independently, H, a protectinggroup, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycle radical, substitutedheterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclicradical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁,N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁),or sulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycleradical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl,substituted C₁-C₁₂ aminoalkyl or a protecting group.
 11. The shortantisense compound of claim 10, wherein each of said bridges is,independently, 4′-CH₂-2′, 4′-(CH₂)₂-2′,4′-CH₂—O-2′,4′-(CH₂)₂—O-2′,4′-CH₂—O—N(R₁)-2′ and 4′-CH₂—N(R₁)—O-2′-wherein each R₁ is, independently, H, a protecting group or C₁-C₁₂alkyl.
 12. The short antisense compound of claim 1, wherein each of thehigh-affinity modified monomer is independently selected from bicyclicnucleotides or other 2′-modified nucleotides.
 13. The short antisensecompound of claim 12, wherein the 2′-modified nucleotides are selectedfrom halogen, allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃,O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)) orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(r) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.
 14. Theshort antisense compound of claim 13, wherein the 2′-modified nucleotideis a 2′-OCH₂CH₂OCH₃ nucleotide.
 15. The short antisense compound ofclaim 1, wherein at least one monomeric linkage is a modified monomericlinkage.
 16. The antisense compound of claim 15, wherein the modifiedmonomeric linkage is a phosphorothioate linkage.
 17. The short antisensecompound of claim 1, wherein each monomeric linkage is aphosphorothioate internucleoside linkage.
 18. The short antisensecompound of any of claims 1-17, that is 8-15 monomers in length.
 19. Theshort antisense compound of claim 18 that is 9-15 monomers in length.20. The short antisense compound of claim 18 that is 10-15 monomers inlength.
 21. The short antisense compound of claim 18 that is 9-14monomers in length.
 22. The short antisense compound of claim 18 that is10-14 monomers in length.
 23. The short antisense compound of claim 18that is 9-13 monomers in length.
 24. The short antisense compound ofclaim 18 that is 10-13 monomers in length.
 25. The short antisensecompound of claim 18 that is 9-12 monomers in length.
 26. The shortantisense compound of claim 18 that is 10-12 monomers in length.
 27. Theshort antisense compound of claim 18 that is 9-11 monomers in length.28. The short antisense compound of claim 18 that is 10-11 monomers inlength.
 29. The short antisense compound of claim 18 that is 8 monomersin length.
 30. The short antisense compound of claim 18 that is 9monomers in length.
 31. The short antisense compound of claim 18 that is10 monomers in length.
 32. The short antisense compound of claim 18 thatis 11 monomers in length.
 33. The short antisense compound of claim 18that is 12 monomers in length.
 34. The short antisense compound of claim18 that is 13 monomers in length.
 35. The short antisense compound ofclaim 18 that is 14 monomers in length.
 36. The short antisense compoundof claim 18 that is 15 monomers in length.
 37. The short antisensecompound of claim 18 that is 16 monomers in length.